Methods for improving milk production by administration of microbial consortia

ABSTRACT

The disclosure relates to isolated microorganisms—including novel strains of the microorganisms—microbial consortia, and compositions comprising the same. Furthermore, the disclosure teaches methods of utilizing the described microorganisms, microbial consortia, and compositions comprising the same, in methods for modulating the production and yield of milk and milk components in ruminants. In particular aspects, the disclosure provides methods of increasing desirable components of milk in ruminants. Furthermore, the disclosure provides for methods of modulating the rumen microbiome.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/276,142, filed Jan. 7, 2016; U.S. ProvisionalApplication No. 62/276,531, filed Jan. 8, 2016; U.S. ProvisionalApplication No. 62/334,816, filed May 11, 2016; and U.S. ProvisionalApplication No. 62/415,908, filed Nov. 1, 2016; each of which is hereinincorporated by reference in its entirety.

FIELD

The present disclosure relates to isolated and biologically puremicroorganisms that have applications, inter alia, in dairy production.The disclosed microorganisms can be utilized in their isolated andbiologically pure states, as well as being formulated into compositions.Furthermore, the disclosure provides microbial consortia, containing atleast two members of the disclosed microorganisms, as well as methods ofutilizing said consortia. Furthermore, the disclosure provides formethods of modulating the rumen microbiome.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is ASBI_002_02US_SeqList_ST25.txt. The text file is893 kb, was created on Oct. 31, 2016, and is being submittedelectronically via EFS-Web.

BACKGROUND

The global population is predicted to increase to over 9 billion peopleby the year 2050 with a concurrent reduction in the quantity of land,water, and other natural resources available per capita. Projectionsindicate that the average domestic income will also increase, with theprojected rise in the GDP of China and India. The desire for a dietricher in animal-source proteins rises in tandem with increasing income,thus the global livestock sector will be charged with the challenge ofproducing more milk using fewer resources. The Food and AgricultureOrganization of the United Nations predict that 70% more food will haveto be produced, yet the area of arable land available will decrease. Itis clear that the food output per unit of resource input will have toincrease considerably in order to support the rise in population.

Milk and milk components from lactating ruminants are predominantlyutilized in the preparation of foodstuffs in many different forms.Nevertheless, milk and milk components find numerous alternativeapplications in non-food areas such as the manufacture of glues, textilefibers, plastic materials, or in the production of ethanol or methane.There have been many strategies to improve milk production and contentin ruminants through nutritional modulations, hormone treatments,changes in animal management, and selective breeding; however, the needfor more efficient production of milk and milk components per animal isrequired.

Identifying compositions and methods for sustainably increasing milkproduction and modulating milk components of interest while balancinganimal health and wellbeing have become imperative to satisfy the needsof every day humans in an expanding population. Increasing the worldwideproduction of milk and further modulating desirable milk components byscaling up the total number of livestock on dairy farms would not onlybe economically infeasible for many parts of the world, but wouldfurther result in negative environmental consequences.

Thus, meeting global milk and milk component yield expectations, bysimply scaling up current high-input agricultural systems—utilized inmost of the developed world—is simply not feasible.

There is therefore an urgent need in the art for improved methods ofincreasing milk production and further increasing yield of desirablemilk components.

SUMMARY OF THE DISCLOSURE

In some aspects, the present disclosure provides isolated microbes,including novel strains of microbes, presented in Table 1 and/or Table3.

In other aspects, the present disclosure provides isolated wholemicrobial cultures of the microbes identified in Table 1 and Table 3.These cultures may comprise microbes at various concentrations.

In some aspects, the disclosure provides for utilizing one or moremicrobes selected from Table 1 and/or Table 3 to increase a phenotypictrait of interest in a ruminant. Furthermore, the disclosure providesfor methods of modulating the rumen microbiome by utilizing one or moremicrobes selected from Table 1 and/or Table 3.

In some embodiments, a microbial consortium comprises at least twomicrobial strains selected from Table 1 and/or Table 3. In someembodiments, a microbial consortium comprises at least one microbialstrain selected from Table 1 and/or Table 3. In a further embodiment, amicrobial consortium comprises at least two microbial strains, whereineach microbe comprise a 16S rRNA sequence encoded by a sequence selectedfrom SEQ ID NOs:1-30 and 2045-2103 or an ITS sequence selected from SEQID NOs:31-60 and 2104-2107. In an additional embodiment, a microbialconsortium comprises at least one microbial strain, wherein each microbecomprise a 16S rRNA sequence encoded by a sequence selected from SEQ IDNOs:1-30 and 2045-2103, or an ITS sequence selected from SEQ IDNOs:31-60 and 2104-2107.

In some embodiments, the microbial consortia of the present disclosurecomprise at least two microbial strains, wherein each microbe comprisesa 16S rRNA sequence encoded by a sequence selected from SEQ ID NOs:1-30,SEQ ID NOs:61-1988, or SEQ ID NOs:2045-2103; or an ITS sequencesselected from SEQ ID NOs:31-60, SEQ ID NOs:1989-2044, or SEQ IDNOs:2104-2107.

In one embodiment, the microbial consortium comprises at least twomicrobial strains comprising Ascusb_7, Ascusb_32, Ascusf_45, andAscusf_24. In a further embodiment, the microbial consortium comprisesat least one microbial strain comprising Ascusb_7, Ascusb_32, Ascusf_45,and Ascusf_24. In one embodiment, the microbial consortium comprises atleast two microbial strains comprising Ascusb_7, Ascusb_32, Ascusf_45,and Ascusf_24. In a further embodiment, the microbial consortiumcomprises at least one microbial strain comprising Ascusb_7, Ascusb_32,Ascusf_45, and Ascusf_24. In one embodiment, the microbial consortiumcomprises at least two microbial strains comprising Ascusb_7,Ascusb_1801, Ascusf_45, and Ascusf_24. In a further embodiment, themicrobial consortium comprises at least one microbial strain comprisingAscusb_7, Ascusb_1801, Ascusf_45, and Ascusf_24. In one embodiment, themicrobial consortium comprises at least two microbial strains comprisingAscusb_7, Ascusb_268, Ascusf_45, and Ascusf_24. In a further embodiment,the microbial consortium comprises at least one microbial straincomprising Ascusb_7, Ascusb_268, Ascusf_45, and Ascusf_24. In oneembodiment, the microbial consortium comprises at least two microbialstrains comprising Ascusb_7, Ascusb_232, Ascusf_45, and Ascusf_24. In afurther embodiment, the microbial consortium comprises at least onemicrobial strain comprising Ascusb_7, Ascusb_232, Ascusf_45, andAscusf_24. In one embodiment, the microbial consortium comprises atleast two microbial strains comprising Ascusb_7, Ascusb_32, Ascusf_45,and Ascusf_249. In a further embodiment, the microbial consortiumcomprises at least one microbial strain comprising Ascusb_7, Ascusb_32,Ascusf_45, and Ascusf_249. In one embodiment, the microbial consortiumcomprises at least two microbial strains comprising Ascusb_7, Ascusb_32,Ascusf_45, and Ascusf_353. In a further embodiment, the microbialconsortium comprises at least one microbial strain comprising Ascusb_7,Ascusb_32, Ascusf_45, and Ascusf_353. In one embodiment, the microbialconsortium comprises at least two microbial strains comprising Ascusb_7,Ascusb_32, Ascusf_45, and Ascusf_23. In a further embodiment, themicrobial consortium comprises at least two microbial strains comprisingAscusb_7, Ascusb_32, Ascusf_45, and Ascusf_23. In one embodiment, themicrobial consortium comprises at least two microbial strains comprisingAscusb_3138 and Ascusf_15. In a further embodiment, the microbialconsortium comprises at least one microbial strain comprisingAscusb_3138 and Ascusf_15. In one embodiment, the at least one microbialstrain comprises Ascusb_3138. In another embodiment, the at least onemicrobial strain comprises Ascusf_15.

In one embodiment, a composition comprises a microbial consortium of thepresent disclosure and an acceptable carrier. In a further embodiment, acomposition comprises a microbial consortium of the present disclosureand acceptable carrier. In a further embodiment, the microbialconsortium is encapsulated. In a further embodiment, the encapsulatedmicrobial consortium comprises a polymer. In a further embodiment, thepolymer may be selected from a saccharide polymer, agar polymer, agarosepolymer, protein polymer, sugar polymer, and lipid polymer.

In some embodiments, the acceptable carrier is selected from the groupconsisting of edible feed grade material, mineral mixture, water,glycol, molasses, and corn oil. In some embodiments, the at least twomicrobial strains forming the microbial consortium are present in thecomposition at 10² to 10¹⁵ cells per gram of said composition.

In some embodiments, the composition may be mixed with livestock feed.

In some embodiments, a method of imparting at least one improved traitupon an animal comprises administering the composition to the animal. Infurther embodiments, the animal is a ruminant, which may further be acow.

In some embodiments, the composition is administered at least once perday. In a further embodiment, the composition is administered at leastonce per month. In a further embodiment, the composition is administeredat least once per week. In a further embodiment, the composition isadministered at least once per hour.

In some embodiments, the administration comprises injection of thecomposition into the rumen. In some embodiments, the composition isadministered anally. In further embodiments, anal administrationcomprises inserting a suppository into the rectum. In some embodiments,the composition is administered orally. In some aspects, the oraladministration comprises administering the composition in combinationwith the animal's feed, water, medicine, or vaccination. In someaspects, the oral administration comprises applying the composition in agel or viscous solution to a body part of the animal, wherein the animalingests the composition by licking. In some embodiments, theadministration comprises spraying the composition onto the animal, andwherein the animal ingests the composition. In some embodiments, theadministration occurs each time the animal is fed. In some embodiments,the oral administration comprises administering the composition incombination with the animal feed.

In some embodiments, the at least one improved trait is selected fromthe group consisting of: an increase of fat in milk, an increase ofcarbohydrates in milk, an increase of protein in milk, an increase ofvitamins in milk, an increase of minerals in milk, an increase in milkvolume, an improved efficiency in feed utilization and digestibility, anincrease in polysaccharide and lignin degradation, an increase in fattyacid concentration in the rumen, pH balance in the rumen, a reduction inmethane emissions, a reduction in manure production, improved dry matterintake, an increase in energy corrected milk (ECM) by weight and/orvolume, an improved efficiency of nitrogen utilization, and anycombination thereof; wherein said increase or reduction is determined bycomparing against an animal not having been administered saidcomposition.

In some embodiments, the increase in fat in milk is an increase intriglycerides, triacylglycerides, diacylglycerides, monoacylglycerides,phospholipids, cholesterol, glycolipids, and/or fatty acids. In someembodiments, an increase of carbohydrates is an increase inoligosaccharides, lactose, glucose, and/or glucose. In some embodiments,an increase in polysaccharide degradation is an increase in thedegradation of cellulose, lignin, and/or hemicellulose. In someembodiments, an increase in fatty acid concentration is an increase inacetic acid, propionic acid, and/or butyric acid.

In some embodiments, the at least two microbial strains or the at leastone microbial strain present in a composition, or consortia, of thedisclosure exhibit an increased utility that is not exhibited when saidstrains occur alone or when said strains are present at a naturallyoccurring concentration. In some embodiments, compositions of thedisclosure, comprising at least two microbial strains as taught herein,exhibit a synergistic effect on imparting at least one improved trait inan animal. In some embodiments, the compositions of thedisclosure—comprising one or more isolated microbes as taughtherein—exhibit markedly different characteristics/properties compared totheir closest naturally occurring counterpart. That is, the compositionsof the disclosure exhibit markedly different functional and/orstructural characteristics/properties, as compared to their closestnaturally occurring counterpart. For instance, the microbes of thedisclosure are structurally different from a microbe as it naturallyexists in a rumen, for at least the following reasons: said microbe canbe isolated and purified, such that it is not found in the milieu of therumen, said microbe can be present at concentrations that do not occurin the rumen, said microbe can be associated with acceptable carriersthat do not occur in the rumen, said microbe can be formulated to beshelf-stable and exist outside the rumen environment, and said microbecan be combined with other microbes at concentrations that do not existin the rumen. Further, the microbes of the disclosure are functionallydifferent from a microbe as it naturally exists in a rumen, for at leastthe following reasons: said microbe when applied in an isolated andpurified form can lead to modulation of the rumen microbiome, increasedmilk production, and/or improved milk compositional characteristics,said microbe can be formulated to be shelf-stable and able to existoutside the rumen environment, such that the microbe now has a newutility as a supplement capable of administration to a ruminant, whereinthe microbe could not have such a utility in it's natural state in therumen, as the microbe would be unable to survive outide the rumenwithout the intervention of the hand of man to formulate the microbeinto a shelf-stable state and impart this new utility that has theaforementioned functional characteristics not possessed by the microbein it's natural state of existence in the rumen.

In one embodiment, the disclosure provides for a ruminant feedsupplement capable of increasing a desirable phenotypic trait in aruminant. In a particular embodiment, the ruminant feed supplementcomprises: a microbial consortium of the present disclosure at aconcentration that does not occur naturally, and an acceptable carrier.In one aspect, the microbial consortium is encapsulated.

In one embodiment, an isolated microbial strain is selected from any oneof the microbial strains in Table 1 and/or Table 3. In one embodiment,an isolated microbial strain is selected from the group consisting of:Ascusb_7 deposited as Bigelow Accession Deposit No. Patent201612011;Ascusb_32 deposited as Bigelow Accession Deposit No. Patent201612007;Ascusb_82 deposited as Bigelow Accession Deposit No. Patent201612012;Ascusb_119 deposited as Bigelow Accession Deposit No. Patent201612009;Ascusb_1801 deposited as Bigelow Accession Deposit No. Patent201612009;Ascusf_206 deposited as Bigelow Accession Deposit No. Patent201612003;Ascusf_23 deposited as Bigelow Accession Deposit No. Patent201612014;Ascusf_24 deposited as Bigelow Accession Deposit No. Patent201612004;Ascusf_45 deposited as Bigelow Accession Deposit No. Patent201612002;Ascusf_208 deposited as Bigelow Accession Deposit No. Patent201612003;Ascusb_3138 deposited as NRRL Accession Deposit No. B-67248; andAscusf_15 deposited as NRRL Accession Deposit No. Y-67249.

In one embodiment, an isolated microbial strain of the presentdisclosure comprises a polynucleotide sequence sharing at least 90%sequence identity with any one of SEQ ID NOs:1-2107. In anotherembodiment, an isolated microbial strain of the present disclosurecomprises a polynucleotide sequence sharing at least 90% sequenceidentity with any one of SEQ ID NOs:1-60 and 2045-2107.

In one embodiment, a substantially pure culture of an isolated microbialstrain may comprise any one of the strains or microbes of the presentdisclosure.

In one embodiment, a method of modulating the microbiome of a ruminantcomprises administering a composition of the present disclosure. In afurther embodiment, the administration of the composition imparts atleast one improved train upon the ruminant. In one embodiment, the atleast one improved trait is selected from the group consisting of: anincrease of fat in milk, an increase of carbohydrates in milk, anincrease of protein in milk, an increase of vitamins in milk, anincrease of minerals in milk, an increase in milk volume, an improvedefficiency in feed utilization and digestibility, an increase inpolysaccharide and lignin degradation, an increase in fatty acidconcentration in the rumen, pH balance in the rumen, a reduction inmethane emissions, a reduction in manure production, improved dry matterintake, an increase in energy corrected milk (ECM) by weight and/orvolume, and an improved efficiency of nitrogen utilization; wherein saidincrease or reduction is determined by comparing against an animal nothaving been administered said composition. In an additional embodiment,the modulation of the microbiome is a decrease in the proportion of themicrobial strains present in the microbiome prior to the administrationof the composition, wherein the decrease is measured relative to themicrobiome of the ruminant prior to the administration of thecomposition.

In one embodiment, the method of increasing fat in milk is an increasein triglycerides, triacylglycerides, diacylglycerides,monoacylglycerides, phospholipids, cholesterol, glycolipids, and/orfatty acids.

In one embodiment, the method of increasing carbohydrates is an increasein oligosaccharides, lactose, glucose, and/or galactose.

In one embodiment, the method of increasing polysaccharide degradationis an increase in the degradation of lignin, cellulose, pectin and/orhemicellulose.

In one embodiment, the method of increasing fatty acid concentration isan increase in acetic acid, propionic acid, and/or butyric acid.

In one embodiment, the method of modulation of the microbiome is anincrease in the proportion of the at least one microbial strain of themicrobiome, wherein the increase is measured relative to a ruminant thatdid not have the at least one microbial strain administered.

In one embodiment, the method of modulation of the microbiome is adecrease in the proportion of the microbial strains present in themicrobiome prior to the administration of the composition, wherein thedecrease is measured relative to the microbiome of the ruminant prior tothe administration of the composition.

In one embodiment, a method of increasing resistance of cows to thecolonization of pathogenic microbes comprises administering acomposition of the present disclosure, resulting in the pathogenicmicrobes being unable to colonize the gastrointestinal tract of a cow.In another embodiment, a method for treating cows for the presence of atleast one pathogenic microbe comprises the administration of a microbialconsortium of the present disclosure and an acceptable carrier. In afurther embodiment, the administration of the microbial consortium ormicrobial composition results in the relative abundance of the at leastone pathogenic microbe to decrease to less than 5% relative abundance inthe gastrointestinal tract. In another embodiment, the administration ofthe microbial consortium or microbial composition results in therelative abundance of the at least one pathogenic microbe to decrease toless than 1% relative abundance in the gastrointestinal tract. Inanother embodiment, the administration of the microbial consortium ormicrobial composition results in the pathogenic microbe beingundetectable in the gastrointestinal tract.

In one embodiment, the microbial compositions and/or consortium comprisebacteria and/or fungi in spore form. In one embodiment, the microbialcompositions and/or consortium of the disclosure comprise bacteriaand/or fungi in whole cell form. In one embodiment, the microbialcompositions and/or consortium of the disclosure comprise bacteriaand/or fungi in lysed cell form. In some aspects of formulating microbesaccording to the dislcosure, the microbes are:fermented→filtered→centrifuged→lyophilized or spray dried→and optionallycoated (i.e. a “fluidized bed step”).

Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedures

Some microorganisms described in this Application were deposited on Apr.25, 2016¹, with the United States Department of Agriculture (USDA)Agricultural Research Service (ARS) Culture Collection (NRRL®), locatedat 1815 N. University St., Peoria, Ill. 61604, USA. Some microorganismsdescribed in this application were deposited with the Bigelow NationalCenter for Marine Algae and Microbiota, located at 60 Bigelow Drive,East Boothbay, Me. 04544, USA. ¹ ASC-01 (NRRL B-67248) and ASC-02 (NRRLY-67249) were deposited on this date

The deposits were made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. The NRRL® and/or Bigelow National Centerfor Marine Algae and Microbiota accession numbers for the aforementionedBudapest Treaty deposits are provided in Table 3. The accession numbersand corresponding dates of deposit for the microorganisms described inthis Application are separately provided in Table 25.

The strains designated in the below tables have been deposited in thelabs of Ascus Biosciences, Inc. since at least Dec. 15, 2015.

In Table 1, the closest predicted hits for taxonomy of the microbes arelisted in columns 2, and 5. Column 2 is the top taxonomic hit predictedby BLAST, and column 5 is the top taxonomic hit for genus+speciespredicted by BLAST. The strains designated in the below table have beendeposited in the labs of Ascus Biosciences, Inc. since at least Dec. 15,2015.

TABLE 1 Microbes of the present disclosure, including bacteria (1-89)and fungi (90-123). Sequence BLAST Identifier BLAST Blast Taxonomic TopBlast for Predicted Taxa of Taxonomic Top % Query Hit w/Genus + % QueryStrain Associated MIC Isolated Microbes Hit Ident. Cover SpeciesIdentity Cover Designation Marker Score 1. Clostridium IV Clostridiaceae96% 100%  Ruminococcus 91% 82% Ascusb_5 SEQ ID 0.85694 (Cluster)bacterium bromii NO: 1 2. Ruminococcus Rumen bacterium 93% 84%Ruminococcus 91% 82% Ascusb_7 SEQ ID 0.97384 (Genus) bromii NO: 2 3.Clostridium IV Rumen bacterium 89% 97% Intestinimonas 85% 100% Ascusb_26 SEQ ID 0.82051 (Cluster) NK4A214 butyriciproducens NO: 3 4.Roseburia (Genus) Lachno- spiraceae 89% 100%  Pseudobutyrivibrio 89% 96%Ascusb_27 SEQ ID 0.87214 bacterium ruminis NO: 4 5.Hydrogenoanaerobacterium (Genus) Lachno- spiraceae 87% 93% Roseburia 86%93% Ascusb_32 SEQ ID 0.81269 bacterium inulinivorans NO: 5 6.Clostridium XIVa Eubacterium 92% 100%  Eubacterium 92% 100%  Ascusb_79SEQ ID 0.82765 (Cluster) ventriosum ventriosum NO: 6 7.Saccharofermentans Rumen bacterium 87% 100%  Faecalibacterium 91% 76%Ascusb_82 SEQ ID 0.93391 (Genus) prausnitzii NO: 7 8. SaccharofermentansSaccharofermentans 100%  99% Saccharofermentans 83% 92% Ascusb_102 SEQID 0.82247 (Genus) sp. acetigenes NO: 8 9. Butyricicoccus Clostridiumsp. 87% 100%  Ruminococcus 86% 99% Ascusb_89 SEQ ID 0.74361 (Genus)flavefaciens NO: 9 10. Papillibacter Rumen bacterium 91% 99% Clostridium88% 82% Ascusb_111 SEQ ID 0.82772 (Genus) NK4A214 saccharolyticum NO: 1011. Ruminococcus Ruminococcaceae 100%  94% Clostridium 85% 99%Ascusb_119 SEQ ID 0.8263 (Genus) lentocellum NO: 11 12.Hydrogenoanaerobacterium Rumen bacterium 85% 98% Ruminococcus 85% 100% Ascusb_145 SEQ ID 0.81161 (Genus) NK4B29 flavefaciens NO: 12 13.Pelotomaculum Faecalibacterium 86% 93% Faecalibacterium 86% 82%Ascusb_205 SEQ ID 0.81461 (Genus) sp. prausnitzii NO: 13 14.Saccharofermentans Bacterium MA3003 99% 91% Saccharofermentans 90% 79%Ascusb_232 SEQ ID 0.81428 (Genus) acetigenes NO: 14 15. LachnospiraceaeBacterium 95% 93% Blautia luti 88% 92% Ascusb_252 SEQ ID 0.8196 incertaesedis (Family) VCD3003 NO: 15 16. Butyricicoccus Ruminococcaceae 91% 77%Clostridium 83% 99% Ascusb_268 SEQ ID 0.74813 sensu stricto (Genus)bacterium lentocellum NO: 16 17. Lachnospiraceae Bacterium 96% 92%Coprococcus catus 88% 100%  Ascusb_374 SEQ ID 0.76214 incertae sedis(Family) YAB2006 NO: 17 18. Anaeroplasma Anaeroplasma 97% 100% Anaeroplasma 97% 100%  Ascusb_411 SEQ ID 0.76213 (Genus) varium variumNO: 18 19. Clostridium sensu Clostridiales 100%  93% Clostridium 81% 91%Ascusb_546 SEQ ID 0.83869 stricto (Genus) bacterium stercorarium NO: 1920. Butyricicoccus Clostridiales 88% 91% Aminiphilus 80% 77% Ascusb_672SEQ ID 0.74829 (Genus) bacterium circumscriptus NO: 20 21.Butyricicoccus Clostridiales 89% 89% Aminiphilus 97% 27% Ascusb_765 SEQID 0.74111 (Genus) bacterium circumscriptus NO: 21 22. Rikenella (Genus)Bacteroides sp. 93% 64% Alistipes shahii 93% 64% Ascusb_812 SEQ ID0.73874 NO: 22 23. Tannerella (Genus) Alistipes shahii 86% 100% Alistipes shahii 86% 100%  Ascusb_1295 SEQ ID 0.8365 NO: 23 24.Howardella (Genus) Clostridiales 85% 100%  Oscillibacter 89% 41%Ascusb_1763 SEQ ID 0.75083 bacterium valericigenes NO: 24 25. Prevotella(Genus) Bacteroidetes 97% 95% Odoribacter 77% 86% Ascusb_1780 SEQ ID0.89749 bacterium splanchnicus NO: 25 26. Butyricimonas Bacteroidetes95% 99% Tannerella forsythia 83% 92% Ascusb_1801 SEQ ID 0.89664 (Genus)bacterium NO: 26 27. Clostridium sensu Bacterium 96% 93%Hydrogenoanaerobacterium 84% 86% Ascusb_1833 SEQ ID 0.73989 stricto(Genus) XBB3002 saccharovorans NO: 27 28. Clostridium sensu Clostridium98% 100%  Clostridium 98% 100%  Ascusb_3138 SEQ ID 0.76524 stricto(Genus) butyricum butyricum NO: 28 29. Saccharofermentans Rumenbacterium 87% 99% Faecalibacterium 90% 76% Ascusb_6589 SEQ ID 0.76539(Genus) NK4A214 prausnitzii NO: 29 30. Lachnospiraceae Roseburia 90%100%  Roseburia 90% 100%  Ascusb_7921 SEQ ID 0.86201 incertae sedis(Family) intestinalis intestinalis NO: 30 31. SuccinivibrioSuccinivibrio 95% 99% Succinivibrio 95% 99% Ascusb_11 SEQ ID 0.50001(Genus) dextrinosolvens dextrinosolvens NO: 2045 32. Prevotella (Genus)Bacterium 100%  93% Prevotella 91% Ascusb_36 SEQ ID 0.55431 MB2027ruminicola NO: 2046 33. Prevotella (Genus) Prevotella 100%  99%Prevotella 100%  Ascusb_67 SEQ ID 0.49156 ruminicola ruminicola NO: 204734. Prevotella (Genus) Prevotella 97% 100%  Prevotella 97% 100% Ascusb_87 SEQ ID 0.59635 ruminicola ruminicola NO: 2048 35. RuminobacterRuminobacter sp. 92% 99% Ruminobacter 92% 100%  Ascusb_101 SEQ ID0.75099 (Genus) amylophilus NO: 2049 36. Syntrophococcus Blautiaproducta 91% 100%  Blautia producta 91% 100%  Ascusb_104 SEQ ID 0.70044(Genus) NO: 2050 37. Succinivibrio Succinivibrio 96% 99% Succinivibrio96% 99% Ascusb_125 SEQ ID 0.44408 (Genus) dextrinosolvensdextrinosolvens NO: 2051 38. Pseudobutyrivibrio Butyrivibrio 99% 100% Butyrivibrio 99% 100%  Ascusb_149 SEQ ID 0.50676 (Genus) fibrisolvensfibrisolvens NO: 2052 39. Prevotella (Genus) Prevotella 99% 99%Prevotella 99% 99% Ascusb_159 SEQ ID 0.5744 ruminicola ruminicola NO:2053 40. Prevotella (Genus) Prevotella 96% 99% Prevotella 96% 99%Ascusb_183 SEQ ID 0.50204 ruminicola ruminicola NO: 2054 41. Prevotella(Genus) Prevotella 99% 100%  Prevotella 99% 100%  Ascusb_187 SEQ ID0.56688 ruminicola ruminicola NO: 2055 42. Prevotella (Genus) BacteriumXBB2006 100%  94% Prevotella albensis 87% 97% Ascusb_190 SEQ ID 0.56183NO: 2056 43. Lachnospiraceae Lachnospiraceae 91% 100%  Roseburia 89%100%  Ascusb_199 SEQ ID 0.62487 incertae sedis (Family) bacteriuminulinivorans NO: 2057 44. Syntrophococcus Ruminococcus 95% 100% Ruminococcus 95% 100%  Ascusb_278 SEQ ID 0.51235 (Genus) gnavus gnavusNO: 2058 45. Ruminobacter Ruminobacter sp. 100%  99% Ruminobacter 99%100%  Ascusb_329 SEQ ID 0.4754 (Genus) amylophilus NO: 2059 46.Butyrivibrio Butyrivibrio sp. 100%  100%  Butyrivibrio 99% 98%Ascusb_368 SEQ ID 0.60727 (Genus) hungatei NO: 2060 47. Clostridium_XIVaEubacterium 100%  96% Eubacterium 100%  96% Ascusb_469 SEQ ID 0.66345(Cluster) oxidoreducens oxidoreducens NO: 2061 48. Prevotella (Genus)Rumen bacterium 99% 99% Prevotella brevis 91% 100%  Ascusb_530 SEQ ID0.44804 NK4A111 NO: 2062 49. Prevotella (Genus) Prevotella sp. 100%  93%Prevotella copri 100%  93% Ascusb_728 SEQ ID 0.55431 NO: 2063 50.Lachnospiraceae Eubacterium 99% 100%  Eubacterium 99% 100%  Ascusb_756SEQ ID 0.72136 incertae sedis (Family) ruminantium ruminantium NO: 206451. Roseburia (Genus) Lachnospiraceae 89% 93% [Clostridium] 89% 91%Ascusb_810 SEQ ID 0.65527 bacterium xylanovorans NO: 2065 52.Lachnospiraceae Lachnospira 99% 100%  Lachnospira 99% 100%  Ascusb_817SEQ ID 0.46512 incertae sedis (Family) pectinoschiza pectinoschiza NO:2066 53. Butyrivibrio Butyrivibrio 98% 99% Butyrivibrio 98% 99%Ascusb_826 SEQ ID 0.65357 (Genus) fibrisolvens fibrisolvens NO: 2067 54.Pseudobutyrivibrio Pseudobutyrivibrio 100%  95% Pseudobutyrivibrio 97%100%  Ascusb_880 SEQ ID 0.52295 (Genus) sp. ruminis NO: 2068 55.Turicibacter Sinimarinibacterium 87% 69% Sinimarinibacterium 87% 69%Ascusb_913 SEQ ID 0.55141 (Genus) flocculans flocculans NO: 2069 56.Lachnospiraceae Bacterium FB3002 100%  91% Butyrivibrio 90% 100% Ascusb_974 SEQ ID 0.53487 incertae sedis (Family) fibrisolvens NO: 207057. Pseudobutyrivibrio Pseudobutyrivibrio 97% 99% Pseudobutyrivibrio 97%99% Ascusb_1069 SEQ ID 0.55299 (Genus) ruminis ruminis NO: 2071 58.Anaerolinea Chloroflexi 88% 99% Anaerolinea 90% 57% Ascusb_1074 SEQ ID0.50893 (Genus) bacterium thermophila NO: 2072 59. Roseburia (Genus)Lachnospiraceae 98% 99% Eubacterium rectale 94% 100%  Ascusb_1293 SEQ ID0.61745 NO: 2073 60. Propionibacterium Propionibacterium 100%  100% Propionibacterium 100%  100%  Ascusb_1367 SEQ ID 0.54046 (Genus) acnesacnes NO: 2074 61. Clostridium_XIVa Lachnospiraceae 88% 100% Pseudobutyrivibrio 86% 97% Ascusb_1632 SEQ ID 0.46826 (Cluster)bacterium ruminis NO: 2075 62. Olsenella (Genus) Coriobacteriaceae 98%100%  Olsenella profusa 97% 100%  Ascusb_1674 SEQ ID 0.51533 bacteriumNO: 2076 63. Streptococcus Streptococcus 95% 82% Streptococcus 95% 82%Ascusb_1786 SEQ ID 0.48678 (Genus) dentirousetti dentirousetti NO: 207764. Clostridium_XIVa Butyrivibrio sp. 99% 96% Butyrivibrio 93% 100% Ascusb_1812 SEQ ID 0.64367 (Cluster) proteoclasticus NO: 2078 65.Clostridium_XIVa Bacterium 99% 91% Butyrivibrio 96% 99% Ascusb_1850 SEQID 0.57807 (Cluster) DAZ2002 hungatei NO: 2079 66. Roseburia (Genus)Lachnospiraceae 95% 99% Eubacterium 89% 100%  Ascusb_1879 SEQ ID 0.45014bacterium oxidoreducens NO: 2080 67. Clostridium_IV Ruminococcaceae 87%99% Ruminococcus 85% 91% Ascusb_2090 SEQ ID 0.75266 (Cluster) bacteriumbromii NO: 2081 68. Clostridium_XICa Bacterium MA2020 99% 99%Clostridium 85% 90% Ascusb_2124 SEQ ID 0.4673 (Cluster)algidixylanolyticum NO: 2082 69. Lachnospiracea Bacterium YSB2008 94%94% Eubacterium 91% 100%  Ascusb_2198 SEQ ID 0.55249 incertae sedis(Family) ruminantium NO: 2083 70. Erysipelotrichaceae Catenisphaera 90%91% Catenisphaera 90% 91% Ascusb_2511 SEQ ID 0.50619 incertae sedis(Family) adipataccumulans adipataccumulans NO: 2084 71. SolobacteriumErysipelotrichaceae 92% 99% Solobacterium 91% 100%  Ascusb_2530 SEQ ID0.53735 (Genus) bacterium moorei NO: 2085 72. LachnospiraceaeEubacterium 95% 100%  Eubacterium 95% 100%  Ascusb_2597 SEQ ID 0.52028incertae sedis (Genus) ruminantium ruminantium NO: 2086 73. ClostridiumXIVa Butyrivibrio 99% 100%  Butyrivibrio 99% 100%  Ascusb_2624 SEQ ID0.55465 (Cluster) proteoclasticus proteoclasticus NO: 2087 74. Ralstonia(Genus) Ralstonia sp. 94 100%  99% Ralsonia insidiosa 99% 100% Ascusb_2667 SEQ ID 0.52371 NO: 2088 75. Clostridium_XIVa Butyrivibriosp. 97% 94% Butyrivibrio 95% 100%  Ascusb_2836 SEQ ID 0.43374 (Cluster)proteoclasticus NO: 2089 76. Eubacterium Eubacteriaceae 84% 100% Casaltella 87% 82% Ascusb_3003 SEQ ID 0.56301 (Genus) bacteriummassiliensis NO: 2090 77. Lachnobacterium Rumen bacterium 89% 98%Eubacterium 90% 91% Ascusb_3504 SEQ ID 0.52856 (Genus) xylanophilum NO:2091 78. Acholeplasma Acholeplasma 86% 72% Acholeplasma 86% 72%Ascusb_3881 SEQ ID 0.4402 (Genus) brassicae brassicae NO: 2092 79.Selenomonas Mitsuokella 91% 97% Mitsuokella 91% 97% Ascusb_4728 SEQ ID0.4761 (Genus) jalaludinii jalaludinii NO: 2093 80. Prevotella (Genus)Prevotella 98% 100%  Prevotella 98% 100%  Ascusb_4934 SEQ ID 0.56204ruminicola ruminicola NO: 2094 81. Clostridium_XIVa Butyrivibrio sp. 99%99% Butyrivibrio 97% 100%  Ascusb_4959 SEQ ID 0.42892 (Cluster)fibrisolvens NO: 2095 82. Succinivibrio Succinivibrio 86% 84%Succinivibrio 86% 84% Ascusb_5525 SEQ ID 0.51758 (Genus) dextrinosolvensdextrinosolvens NO: 2096 83. Ruminobacter Ruminobacter sp. 100%  99%Ruminobacter 99% 100%  Ascusb_12103 SEQ ID 0.52909 (Genus) amylophilusNO: 2097 84. Sharpea (Genus) Lachnospiraceae 97% 100%  Sharpeaazabuensis 100%  91% Ascusb_14245 SEQ ID 0.61391 bacterium NO: 2098 85.Prevotella (Genus) Prevotella 87% 97% Prevotella 87% 97% Ascusb_14945SEQ ID 0.80101 ruminicola ruminicola NO: 2099 86. Prevotella (Genus)Prevotella sp. DJF 88% 89% Prevotella 87% 945 Ascusb_17461 SEQ ID0.44777 ruminicola NO: 2100 87. Prevotella (Genus) Bacterium MB2027100%  93% Prevotella 91% 99% Ascusb_20083 SEQ ID 0.52538 ruminicola NO:2101 88. Prevotella (Genus) Prevotella 99% 100%  Prevotella 99% 100% Ascusb_20187 SEQ ID 0.59156 ruminicola ruminicola NO: 2102 89.Prevotella (Genus) Prevotella 100%  100%  Prevotella 100%  100% Ascusb_20539 SEQ ID 0.4912 ruminicola ruminicola NO: 2103 90. Piromyces(Genus) Piromyces sp. 93% 100%  Neocallimastix 84% 100%  Ascusf_11 SEQID 0.81719 frontalis NO: 31 91. Candida xylopsoc Pichia kudriavzevii100%  100%  Pichia kudriavzevii 100%  100%  Ascusf_15 SEQ ID 0.76088(Genus + Species) NO: 32 92. Orpinomyces Orpinomyces sp. 100%  100% Neocallimastix 86% 100%  Ascusf_22 SEQ ID 0.76806 (Genus) frontalis NO:33 93. Orpinomycs Neocallimastix 86% 80% Neocallimastix 86% 80%Ascusf_23 SEQ ID 0.85707 (Genus) frontalis frontalis NO: 34 94.Orpinomyces Orpinomyces sp. 95% 100%  Neocallimastix 86% 100%  Ascusf_24SEQ ID 0.85292 (Genus) frontalis NO: 35 95. Candida apicol Candidaapicola 100%  100%  Candida apicola 100%  100%  Ascusf_25 SEQ ID 0.70561(Genus + Species) NO: 36 96. Candida rugosa Candida 100%  100%  Candida100%  100%  Ascusf_38 SEQ ID 0.78246 (Genus + Species) akabanensisakabanensis NO: 37 97. Neocallimastix Neocallimastix sp. 99% 100% Neocallimastix 99% 100%  Ascusf_45 SEQ ID 0.86185 (Genus) frontalis NO:38 98. Orpinomyces Orpinomyces sp. 99% 100%  Orpinomyces 96% 96%Ascusf_60 SEQ ID 0.72985 (Genus) joyonii NO: 39 99. OrpinomycesNeocallimastix 86% 78% Neocallimastix 86% 78% Ascusf_73 SEQ ID 0.76064(Genus) frontalis frontalis NO: 40 100. Neocallimastix Neocallimastixsp. 98% 100%  Neocallimastix 93% 100%  Ascusf_77 SEQ ID 0.83475 (Genus)frontalis NO: 41 101. Neocallimastix Neocallimastix 97% 100% Neocallimastix 97% 100%  Ascusf_94 SEQ ID 0.77644 (Genus) frontalisfrontalis NO: 42 102. Ascomycota (Genus) Basidiomycota sp. 85% 98%Sugiyamaella 97% 26% Ascusf_95 SEQ ID 0.7089 lignohabitans NO: 43 103.Piromyces (Genus) Caecomyces sp. 94% 100%  Cyllamyces 86% 89% Ascusf_108SEQ ID 0.68405 aberensis NO: 44 104. Orpinomyces Orpinomyces sp. 95%100%  Orpinomyces 87% 96% Ascusf_119 SEQ ID 0.80055 (Genus) joyonii NO:45 105. Cyllamyces (Genus) Caecomyces sp. 90% 100%  Caecomyces 90% 83%Ascusf_127 SEQ ID 0.66812 communis NO: 46 106. Piromyces (Genus)Caecomyces sp. 91% 100%  Caecomyces 92% 83% Ascusf_136 SEQ ID 0.73201communis NO: 47 107. Cyllamyces (Genus) Cyllamyces sp. 97% 100% Cyllamyces 94% 89% Ascusf_193 SEQ ID 0.7586 aberensis NO: 48 108.Piromyces (Genus) Piromyces sp. 92% 100%  Neocallimastix 84% 100% Ascusf_228 SEQ ID 0.83403 frontalis NO: 49 109. Piromyces (Genus)Caecomyces sp. 94% 100%  Cyllamyces 86% 89% Ascusf_249 SEQ ID 0.78679aberensis NO: 50 110. Neocallimastix Neocallimastix sp. 98% 100% Neocallimastix 92% 100%  Ascusf_307 SEQ ID 0.77859 (Genus) frontalis NO:51 111. Piromyces (Genus) Piromyces sp. 94% 100%  Neocallimastix 83%100%  Ascusf_315 SEQ ID 0.81028 frontalis NO: 52 112. NeocallimastixNeocallimastix sp. 100%  98% Neocallimastix 100%  90% Ascusf_334 SEQ ID0.76456 (Genus) frontalis NO: 53 113. Saccharomycetales Candidaethanolica 100%  100%  Candida ethanolica 100%  100%  Ascusf_353 SEQ ID0.82628 (Order) NO: 54 114. Piromyces (Genus) Piromyces sp. 91% 100% Neocallimastix 83% 100%  Ascusf_448 SEQ ID 0.70021 frontalis NO: 55 115.Orpinomyces Neocallimastix sp. 88% 91% Neocallimastix 96% 88% Ascusf_786SEQ ID 0.63201 (Genus) frontalis NO: 56 116. Piromyces (Genus) Piromycessp. 91% 100%  Neocallimastix 83% 100%  Ascusf_836 SEQ ID 0.65492frontalis NO: 57 117. Phyllosticta Tremellales sp. 96% 74% Tremellagiraffa 83% 96% Ascusf_923 SEQ ID 0.76115 capitalensis (Genus + NO: 58Species) 118. Orpinomyces Neocallimastix 87% 77% Neocallimastix 87% 77%Ascusf_1020 SEQ ID 0.68043 (Genus) frontalis frontalis NO: 59 119.Orpinomyces Neocallimastix 85% 80% Neocallimastix 85% 80% Ascusf_1103SEQ ID 0.73004 (Genus) frontalis frontalis NO: 60 120. OrpinomycesFungal sp. Tianzhu- 99% 100%  Orpinomyces 94% 96% Ascusf_81 SEQ ID0.44471 (Genus) Yak6 joyonii NO: 2104 121. Piromyces (Genus) Piromycessp. 99% 100%  Neocallimastix 84% 100%  Ascusf_206 SEQ ID 0.49752frontalis NO: 2105 122. Piromyces (Genus) Piromyces sp. 96% 100% Neocallimastix 82% 100%  Ascusf_208 SEQ ID 0.4176 frontalis NO: 2106123. Piromyces (Genus) Piromyces sp. 99% 100%  Neocallimastix 82% 100% Ascusf_1012 SEQ ID 0.55922 frontalis NO: 2107

TABLE 2 Microbial Deposits Corresponding to the Microbes of Table 1Sequence Sequence Identifier Identifier Predicted Taxa for PredictedTaxa for of Isolated Strain Associated of Isolated Strain AssociatedMicrobes Designation Marker Deposit # Microbes Designation MarkerDeposit # Clostridium IV (Cluster) Ascusb_5 SEQ ID PATENT201612001,Streptococcus Ascusb_1786 SEQ ID PATENT201612011, NO: 1 PATENT201612007,(Genus) NO: 2077 PATENT201612012, PATENT201612009, PATENT201612013PATENT201612010, PATENT201612011, PATENT201612012 Ruminococcus (Genus)Ascusb_7 SEQ ID PATENT201612005, Clostridium_XIVa Ascusb_1812 SEQ IDPATENT201612011, NO: 2 PATENT201612007, (Cluster) NO: 2078PATENT201612012 PATENT201612009, PATENT201612010, PATENT201612011,PATENT201612012, PATENT201612013 Clostridium IV (Cluster) Ascusb_26 SEQID PATENT201612005, Clostridium_XIVa Ascusb_1850 SEQ ID PATENT201612013NO: 3 PATENT201612009, (Cluster) NO: 2079 PATENT201612011,PATENT201612012 Roseburia (Genus) Ascusb_27 SEQ ID PATENT201612009Roseburia (Genus) Ascusb_1879 SEQ ID NO: 4 NO: 2080Hydrogenoanaerobacterium Ascusb_32 SEQ ID PATENT201612006,Clostridium_IV Ascusb_2090 SEQ ID PATENT201612007, (Genus) NO: 5PATENT201612009, (Cluster) NO: 2081 PATENT201612009 PATENT201612012Clostridium XIVa Ascusb_79 SEQ ID PATENT201612011, Clostridium_XICaAscusb_2124 SEQ ID PATENT201612012 (Cluster) NO: 6 PATENT201612012(Cluster) NO: 2082 Saccharofermentans Ascusb_82 SEQ ID PATENT201612005,Lachnospiracea Ascusb_2198 SEQ ID PATENT201612012 (Genus) NO: 7PATENT201612006, incertae sedis NO: 2083 PATENT201612007, (Family)PATENT201612009, PATENT201612010, PATENT201612012 SaccharofermentansAscusb_102 SEQ ID PATENT201612005, Erysipelotrichaceae Ascusb_2511 SEQID PATENT201612001, (Genus) NO: 8 PATENT201612007, incertae sedis NO:2084 PATENT201612007, PATENT201612010, (Family) PATENT201612009PATENT201612011, PATENT201612012 Butyricicoccus (Genus) Ascusb_89 SEQ IDPATENT201612011, Solobacterium Ascusb_2530 SEQ ID PATENT201612011, NO: 9PATENT201612012 (Genus) NO: 2085 PATENT201612012 Papillibacter (Genus)Ascusb_111 SEQ ID PATENT201612005, Lachnospiraceae Ascusb_2597 SEQ IDPATENT201612013 NO: 10 PATENT201612007, incertae sedis NO: 2086PATENT201612012 (Genus) Ruminococcus (Genus) Ascusb_119 SEQ IDPATENT201612011, Clostridium_XIVa Ascusb_2624 SEQ ID PATENT201612009,NO: 11 PATENT201612012 (Cluster) NO: 2087 PATENT201612011,PATENT201612012 Hydrogenoanaerobacterium Ascusb_145 SEQ IDPATENT201612011, Ralstonia (Genus) Ascusb_2667 SEQ ID PATENT201612013(Genus) NO: 12 PATENT201612012 NO: 2088 Pelotomaculum (Genus) Ascusb_205SEQ ID PATENT201612005, Clostridium_XIVa Ascusb_2836 SEQ IDPATENT201612013 NO: 13 PATENT201612006, (Cluster) NO: 2089PATENT201612011, PATENT201612012 Saccharofermentans Ascusb_232 SEQ IDPATENT201612010, Eubacterium Ascusb_3003 SEQ ID PATENT201612009 (Genus)NO: 14 PATENT201612011, (Genus) NO: 2090 PATENT201612012 Lachnospiraceaeincertae Ascusb_252 SEQ ID Lachnobacterium Ascusb_3504 SEQ IDPATENT201612011, sedis (Family) NO: 15 (Genus) NO: 2091 PATENT201612012Butyricicoccus sensu Ascusb_268 SEQ ID PATENT201612007, AcholeplasmaAscusb_3881 SEQ ID PATENT201612007 stricto (Genus) NO: 16PATENT201612011, (Genus) NO: 2092 PATENT201612012 Lachnospiraceaeincertae Ascusb_374 SEQ ID PATENT201612007, Selenomonas Ascusb_4728 SEQID sedis (Family) NO: 17 PATENT201612009, (Genus) NO: 2093PATENT201612010, PATENT201612011 PATENT201612012 Anaeroplasma (Genus)Ascusb_411 SEQ ID PATENT201612007, Prevotella (Genus) Ascusb_4934 SEQ IDNO: 18 PATENT201612011, NO: 2094 PATENT201612012 Clostridium sensustricto Ascusb_546 SEQ ID PATENT201612013 Clostridium_XIVa Ascusb_4959SEQ ID (Genus) NO: 19 (Cluster) NO: 2095 Butyricicoccus (Genus)Ascusb_672 SEQ ID Succinivibrio Ascusb_5525 SEQ ID NO: 20 (Genus) NO:2096 Butyricicoccus (Genus) Ascusb_765 SEQ ID PATENT201612013Ruminobacter Ascusb_12103 SEQ ID PATENT201612001 NO: 21 (Genus) NO: 2097Rikenella (Genus) Ascusb_812 SEQ ID PATENT201612005, Sharpea (Genus)Ascusb_14245 SEQ ID PATENT201612001, NO: 22 PATENT201612006, NO: 2098PATENT201612008, PATENT201612011, PATENT201612009, PATENT201612012PATENT201612011, PATENT201612012, PATENT201612013 Tannerella (Genus)Ascusb_1295 SEQ ID PATENT201612007, Prevotella (Genus) Ascusb_14945 SEQID NO: 23 PATENT201612009, NO: 2099 PATENT201612011, PATENT201612012Howardella (Genus) Ascusb_1763 SEQ ID PATENT201612011, Prevotella(Genus) Ascusb_17461 SEQ ID NO: 24 PATENT201612012 NO: 2100 Prevotella(Genus) Ascusb_1780 SEQ ID PATENT201612013 Prevotella (Genus)Ascusb_20083 SEQ ID PATENT201612006 NO: 25 NO: 2101 Butyricimonas(Genus) Ascusb_1801 SEQ ID PATENT201612005 Prevotella (Genus)Ascusb_20187 SEQ ID PATENT201612009, NO: 26 NO: 2102 PATENT201612011,PATENT201612012 Clostridium sensu stricto Ascusb_1833 SEQ IDPATENT201612006, Prevotella (Genus) Ascusb_20539 SEQ ID (Genus) NO: 27PATENT201612007, NO: 2103 PATENT201612009, PATENT201612010,PATENT201612011, PATENT201612012 Clostridium sensu stricto Ascusb_3138SEQ ID PATENT201612005, Piromyces (Genus) Ascusf_11 SEQ ID (Genus) NO:28 PATENT201612006, NO: 31 PATENT201612008, PATENT201612009,PATENT201612010, PATENT201612011, PATENT201612012, PATENT201612013, NRRLB-67248 Saccharofermentans Ascusb_6589 SEQ ID PATENT201612005 Candidaxylopsoc Ascusf_15 SEQ ID NRRL Y-67249, (Genus) NO: 29 (Genus + Species)NO: 32 PATENT201612014 Lachnospiraceae incertae Ascusb_7921 SEQ IDOrpinomyces Ascusf_22 SEQ ID PATENT201612002, sedis (Family) NO: 30(Genus) NO: 33 PATENT201612004 Succinivibrio (Genus) Ascusb_11 SEQ IDPATENT201612001, Orpinomycs Ascusf_23 SEQ ID PATENT201612014 NO: 2045PATENT201612008, (Genus) NO: 34 PATENT201612009, PATENT201612011,PATENT201612012 Prevotella (Genus) Ascusb_36 SEQ ID PATENT201612013Orpinomyces Ascusf_24 SEQ ID PATENT201612002, NO: 2046 (Genus) NO: 35PATENT201612004 Prevotella (Genus) Ascusb_67 SEQ ID Candida apicolAscusf_25 SEQ ID PATENT201612014 NO: 2047 (Genus + Species) NO: 36Prevotella (Genus) Ascusb_87 SEQ ID Candida rugosa Ascusf_38 SEQ IDPATENT201612004 NO: 2048 (Genus + Species) NO: 37 Ruminobacter (Genus)Ascusb_101 SEQ ID PATENT201612001, Neocallimastix Ascusf_45 SEQ IDPATENT201612002, NO: 2049 PATENT201612005, (Genus) NO: 38PATENT201612014 PATENT201612011, PATENT201612012 Syntrophococcus (Genus)Ascusb_104 SEQ ID PATENT201612005, Orpinomyces Ascusf_60 SEQ ID NO: 2050PATENT201612006 (Genus) NO: 39 Succinivibrio (Genus) Ascusb_125 SEQ IDPATENT201612001, Orpinomyces Ascusf_73 SEQ ID NO: 2051 PATENT201612005,(Genus) NO: 40 PATENT201612006, PATENT201612008, PATENT201612009,PATENT201612011, PATENT201612012 Pseudobutyrivibrio Ascusb_149 SEQ IDPATENT201612001, Neocallimastix Ascusf_77 SEQ ID PATENT201612014 (Genus)NO: 2052 PATENT201612008, (Genus) NO: 41 PATENT201612009,PATENT201612011, PATENT201612012, PATENT201612013 Prevotella (Genus)Ascusb_159 SEQ ID PATENT201612005, Neocallimastix Ascusf_94 SEQ IDPATENT201612014 NO: 2053 PATENT201612006, (Genus) NO: 42PATENT201612007, PATENT201612008, PATENT201612009, PATENT201612010,PATENT201612011, PATENT201612012 Prevotella (Genus) Ascusb_183 SEQ IDPATENT201612008, Ascomycota Ascusf_95 SEQ ID NO: 2054 PATENT201612009(Genus) NO: 43 Prevotella (Genus) Ascusb_187 SEQ ID PATENT201612007,Piromyces (Genus) Ascusf_108 SEQ ID PATENT201612014 NO: 2055PATENT201612008, NO: 44 PATENT201612010, PATENT201612011,PATENT201612012 Prevotella (Genus) Ascusb_190 SEQ ID PATENT201612005,Orpinomyces Ascusf_119 SEQ ID NO: 2056 PATENT201612006, (Genus) NO: 45PATENT201612007, PATENT201612012 Lachnospiraceae incertae Ascusb_199 SEQID PATENT201612011, Cyllamyces Ascusf_127 SEQ ID sedis (Family) NO: 2057PATENT201612012 (Genus) NO: 46 Syntrophococcus (Genus) Ascusb_278 SEQ IDPATENT201612008 Piromyces (Genus) Ascusf_136 SEQ ID NO: 2058 NO: 47Ruminobacter (Genus) Ascusb_329 SEQ ID PATENT201612010 CyllamycesAscusf_193 SEQ ID NO: 2059 (Genus) NO: 48 Butyrivibrio (Genus)Ascusb_368 SEQ ID PATENT201612011, Piromyces (Genus) Ascusf_228 SEQ IDNO: 2060 PATENT201612012 NO: 49 Clostridium_XIVa Ascusb_469 SEQ IDPiromyces (Genus) Ascusf_249 SEQ ID (Cluster) NO: 2061 NO: 50 Prevotella(Genus) Ascusb_530 SEQ ID Neocallimastix Ascusf_307 SEQ IDPATENT201612002, NO: 2062 (Genus) NO: 51 PATENT201612014 Prevotella(Genus) Ascusb_728 SEQ ID PATENT201612008, Piromyces (Genus) Ascusf_315SEQ ID NO: 2063 PATENT201612009, NO: 52 PATENT201612011,PATENT201612012, PATENT201612013 Lachnospiraceae incertae Ascusb_756 SEQID Neocallimastix Ascusf_334 SEQ ID PATENT201612014 sedis (Family) NO:2064 (Genus) NO: 53 Roseburia (Genus) Ascusb_810 SEQ ID PATENT201612011,Saccharomycetales Ascusf_353 SEQ ID PATENT201612014 NO: 2065PATENT201612012 (Order) NO: 54 Lachnospiraceae incertae Ascusb_817 SEQID PATENT201612001, Piromyces (Genus) Ascusf_448 SEQ ID sedis (Family)NO: 2066 PATENT201612006, NO: 55 PATENT201612009, PATENT201612012,PATENT201612013, NRRL B-67349 Butyrivibrio (Genus) Ascusb_826 SEQ IDPATENT201612011, Orpinomyces Ascusf_786 SEQ ID NO: 2067 PATENT201612012,(Genus) NO: 56 PATENT201612013, NRRL B-67347 PseudobutyrivibrioAscusb_880 SEQ ID PATENT201612008, Piromyces (Genus) Ascusf_836 SEQ ID(Genus) NO: 2068 PATENT201612009 NO: 57 Turicibacter (Genus) Ascusb_913SEQ ID PATENT201612007, Phyllosticta Ascusf_923 SEQ ID NO: 2069PATENT201612008, capitalensis (Genus + NO: 58 PATENT201612009, Species)PATENT201612010, PATENT201612011, PATENT201612012 Lachnospiraceaeincertae Ascusb_974 SEQ ID PATENT201612013 Orpinomyces Ascusf_1020 SEQID sedis (Family) NO: 2070 (Genus) NO: 59 Pseudobutyrivibrio Ascusb_1069SEQ ID PATENT201612011, Orpinomyces Ascusf_1103 SEQ ID (Genus) NO: 2071PATENT201612012, (Genus) NO: 60 NRRL B-67348 Anaerolinea (Genus)Ascusb_1074 SEQ ID PATENT201612005, Orpinomyces Ascusf_81 SEQ ID NO:2072 PATENT201612007, (Genus) NO: 2104 PATENT201612008, PATENT201612012Roseburia (Genus) Ascusb_1293 SEQ ID Piromyces (Genus) Ascusf_206 SEQ IDPATENT201612003 NO: 2073 NO: 2105 Propionibacterium Ascusb_1367 SEQ IDPATENT201612007, Piromyces (Genus) Ascusf_208 SEQ ID PATENT201612003(Genus) NO: 2074 PATENT201612009, NO: 2106 PATENT201612012Clostridium_XIVa Ascusb_1632 SEQ ID PATENT201612011, Piromyces (Genus)Ascusf_1012 SEQ ID PATENT201612003 (Cluster) NO: 2075 PATENT201612012NO: 2107 Olsenella (Genus) Ascusb_1674 SEQ ID PATENT201612001, NO: 2076PATENT201612009

TABLE 3 Bacteria of the present disclosure. Predicted Closest Taxa ofIsolated Strain Sequence Microbes Designation Identifier CorynebacteriumAscusb_3 61 Prevotella Ascusb_50 62 Comamonas Ascusb_90 63Clostridium_XIVa Ascusb_117 64 Hippea Ascusb_171 65 AnaerovoraxAscusb_177 66 Clostridium_XIVa Ascusb_179 67 Rummeliibacillus Ascusb_22468 Clostridium_XIVa Ascusb_234 69 Lachnospiracea_incertae_sedisAscusb_274 70 Prevotella Ascusb_276 71 Anaerovorax Ascusb_293 72Pseudoflavonifractor Ascusb_327 73 Prevotella Ascusb_337 74Clostridium_XIVa Ascusb_357 75 Clostridium_XIVa Ascusb_357 76Coprococcus Ascusb_361 77 Pyramidobacter Ascusb_388 78 SyntrophococcusAscusb_425 79 Prevotella Ascusb_444 80 Clostridium_XIVa Ascusb_456 81Prevotella Ascusb_492 82 Roseburia Ascusb_523 83 Clostridium_XIVaAscusb_526 84 Lachnospiracea_incertae_sedis Ascusb_570 85Clostridium_XIVa Ascusb_584 86 Acidothermus Ascusb_605 87 AdlercreutziaAscusb_606 88 Prevotella Ascusb_617 89 Lachnospiracea_incertae_sedisAscusb_635 90 Proteiniclasticum Ascusb_642 91Lachnospiracea_incertae_sedis Ascusb_647 92 Anaerovorax Ascusb_656 93Prevotella Ascusb_669 94 Bacteroides Ascusb_681 95 Clostridium_IIIAscusb_704 96 Prevotella Ascusb_706 97 Acinetobacter Ascusb_717 98Erysipelothrix Ascusb_752 99 Bacteroides Ascusb_790 100 Clostridium_XIVaAscusb_797 101 Butyrivibrio Ascusb_802 102 Eubacterium Ascusb_805 103Prevotella Ascusb_828 104 Eubacterium Ascusb_890 105 PrevotellaAscusb_909 106 Lachnospiracea_incertae_sedis Ascusb_924 107 CoprococcusAscusb_955 108 Prevotella Ascusb_958 109 Clostridium_XIVa Ascusb_980 110Prevotella Ascusb_982 111 Catonella Ascusb_990 112 MethanobrevibacterAscusb_993 113 Ruminococcus Ascusb_1013 114Lachnospiracea_incertae_sedis Ascusb_1021 115 Coprococcus Ascusb_1033116 Clostridium_XIVa Ascusb_1090 117 Lachnospiracea_incertae_sedisAscusb_1108 118 Prevotella Ascusb_1113 119 Anaerovorax Ascusb_1114 120Asteroleplasma Ascusb_1116 121 Clostridium_XIVa Ascusb_1118 122Caulobacter Ascusb_1123 123 Lachnospiracea_incertae_sedis Ascusb_1128124 Roseburia Ascusb_1152 125 Clostridium_XIVa Ascusb_1166 126Acinetobacter Ascusb_1170 127 Bacteroides Ascusb_1176 128 ErysipelothrixAscusb_1182 129 Coprococcus Ascusb_1199 130 Clostridium_XIVa Ascusb_1201131 Bacteroides Ascusb_1218 132 Coprococcus Ascusb_1239 133 AnaerovoraxAscusb_1269 134 Pseudoflavonifractor Ascusb_1296 135Pseudoflavonifractor Ascusb_1296 136 Prevotella Ascusb_1298 137Lachnospiracea_incertae_sedis Ascusb_1304 138 Roseburia Ascusb_1320 139Prevotella Ascusb_1330 140 Coprococcus Ascusb_955 108 PrevotellaAscusb_958 109 Clostridium _(—) XIVa Ascusb_980 110 PrevotellaAscusb_982 111 Catonella Ascusb_990 112 Methanobrevibacter Ascusb_993113 Ruminococcus Ascusb_1013 114 Lachnospiracea_incertae_sedisAscusb_1021 115 Coprococcus Ascusb_1033 116 Clostridium_XIVa Ascusb_1090117 Lachnospiracea_incertae_sedis Ascusb_1108 118 Prevotella Ascusb_1113119 Anaerovorax Ascusb_1114 120 Asteroleplasma Ascusb_1116 121Clostridium_XIVa Ascusb_1118 122 Caulobacter Ascusb_1123 123Lachnospiracea_incertae_sedis Ascusb_1128 124 Roseburia Ascusb_1152 125Clostridium_XIVa Ascusb_1166 126 Acinetobacter Ascusb_1170 127Bacteroides Ascusb_1176 128 Erysipelothrix Ascusb_1182 129 CoprococcusAscusb_1199 130 Clostridium_XIVa Ascusb_1201 131 Bacteroides Ascusb_1218132 Coprococcus Ascusb_1239 133 Anaerovorax Ascusb_1269 134Pseudoflavonifractor Ascusb_1296 135 Pseudoflavonifractor Ascusb_1296136 Prevotella Ascusb_1298 137 Lachnospiracea_incertae_sedis Ascusb_1304138 Roseburia Ascusb_1320 139 Prevotella Ascusb_1330 140 RuminococcusAscusb_1336 141 Atopobium Ascusb_1341 142 Eubacterium Ascusb_1347 143Robinsoniella Ascusb_1355 144 Neisseria Ascusb_1357 145 RuminococcusAscusb_1362 146 Prevotella Ascusb_1364 147 Slackia Ascusb_1389 148Prevotella Ascusb_1400 149 Clostridium_XIVa Ascusb_1410 150 BacteroidesAscusb_1417 151 Anaerorhabdus Ascusb_1426 152 Bacteroides Ascusb_1433153 Prevotella Ascusb_1439 154 Corynebacterium Ascusb_1440 155 AtopobiumAscusb_1468 156 Streptophyta Ascusb_1473 157 Prevotella Ascusb_1485 158Roseburia Ascusb_1490 159 Prevotella Ascusb_1492 160 PrevotellaAscusb_1528 161 Eubacterium Ascusb_1538 162 Rhodocista Ascusb_1543 163Prevotella Ascusb_1546 164 Clostridium_XIVa Ascusb_1553 165 PrevotellaAscusb_1554 166 Prevotella Ascusb_1571 167 Streptophyta Ascusb_1578 168Ochrobactrum Ascusb_1580 169 Mogibacterium Ascusb_1591 170 AdlercreutziaAscusb_1600 171 Prevotella Ascusb_1609 172 Riemerella Ascusb_1627 173Prevotella Ascusb_1640 174 Roseburia Ascusb_1645 175 Slackia Ascusb_1647176 Clostridium_IV Ascusb_1656 177 Syntrophococcus Ascusb_1659 178Prevotella Ascusb_1667 179 Treponema Ascusb_1689 180 PrevotellaAscusb_1708 181 Anaerovorax Ascusb_1723 182 Prevotella Ascusb_1727 183Methanobrevibacter Ascusb_1739 184 Corynebacterium Ascusb_1773 185Clostridium_XIVa Ascusb_1793 186 Alkaliphilus Ascusb_1795 187Ruminococcus Ascusb_1797 188 Clostridium_XIVa Ascusb_1806 189Eubacterium Ascusb_1819 190 Bacteroides Ascusb_1835 191 RoseburiaAscusb_1886 192 Lentisphaera Ascusb_1901 193 Eubacterium Ascusb_1905 194Roseburia Ascusb_1918 195 Clostridium_IV Ascusb_1922 196 HahellaAscusb_1947 197 Butyricicoccus Ascusb_1969 198 Clostridium _(—) IVAscusb_2016 199 Prevotella Ascusb_2024 200 Clostridium_IV Ascusb_2058201 Desulfovibrio Ascusb_2081 202 Sphingobacterium Ascusb_2101 203Roseburia Ascusb_2105 204 Bacteroides Ascusb_2131 205 RuminococcusAscusb_2141 206 Prevotella Ascusb_2156 207 Asteroleplasma Ascusb_2168208 Syntrophococcus Ascusb_2182 209 Victivallis Ascusb_2199 210Lachnobacterium Ascusb_2210 211 Lachnospiracea_incertae_sedisAscusb_2211 212 Clostridium_IV Ascusb_2218 213 Anaerorhabdus Ascusb_2221214 Altererythrobacter Ascusb_2236 215 Clostridium_XIVa Ascusb_2246 216Clostridium_XIVa Ascusb_2263 217 Proteiniclasticum Ascusb_2264 218Bifidobacterium Ascusb_2308 219 Clostridium_XIVa Ascusb_2322 220Clostridium_XIVa Ascusb_2323 221 Desulfovibrio Ascusb_2332 222Clostridium_XIVa Ascusb_2353 223 Nitrobacter Ascusb_2375 224Enterorhabdus Ascusb_2414 225 Clostridium_sensu_stricto Ascusb_2429 226Oscillibacter Ascusb_2435 227 Nautilia Ascusb_2437 228 CorynebacteriumAscusb_2447 229 Ruminococcus Ascusb_2452 230 Coprococcus Ascusb_2461 231Eubacterium Ascusb_2462 232 Rikenella Ascusb_2470 233 Clostridium_XIVaAscusb_2482 234 Paenibacillus Ascusb_2487 235 Ruminococcus Ascusb_2492236 Prevotella Ascusb_2503 237 Haematobacter Ascusb_2504 238 PrevotellaAscusb_2523 239 Clostridium_XIVa Ascusb_2537 240Lachnospiracea_incertae_sedis Ascusb_2538 241 Enterorhabdus Ascusb_2565242 Blautia Ascusb_2591 243 Sporobacter Ascusb_2592 244 OscillibacterAscusb_2607 245 Clostridium_XIVa Ascusb_2608 246 Atopobium Ascusb_2613247 Sporobacter Ascusb_2626 248 Clostridium_XIVa Ascusb_2629 249Candidate Phylum OD1 Ascusb_2643 250 Oscillibacter Ascusb_2645 251Clostridium_XIVa Ascusb_2647 252 Clostridium_IV Ascusb_2649 253Mogibacterium Ascusb_2653 254 Roseburia Ascusb_2663 255Lachnospiracea_incertae_sedis Ascusb_2671 256 Pelotomaculum Ascusb_2696257 Pelotomaculum Ascusb_2712 258 Clostridium_XIVa Ascusb_2713 259Robinsoniella Ascusb_2730 260 Coprococcus Ascusb_2746 261 WautersiellaAscusb_2757 262 Lachnospiracea_incertae_sedis Ascusb_2762 263Planctomyces Ascusb_2764 264 Treponema Ascusb_2800 265 CoprococcusAscusb_2806 266 Paracoccus Ascusb_2809 267 Ruminococcus Ascusb_2811 268Atopobium Ascusb_2814 269 Prevotella Ascusb_2825 270 Clostridium_IVAscusb_2832 271 Clostridium_XIVa Ascusb_2838 272 Clostridium_XIVaAscusb_2843 273 Clostridium_XIVa Ascusb_2853 274 Prevotella Ascusb_2857275 Dethiosulfovibrio Ascusb_2872 276 Clostridium_XI Ascusb_2885 277Clostridium_IV Ascusb_2907 278 Saccharofermentans Ascusb_2909 279Clostridium_sensu_stricto Ascusb_2912 280 Roseburia Ascusb_2914 281Lachnospiracea_incertae_sedis Ascusb_2930 282 Candidate phylum SR1Ascusb_2946 283 Hydrogenoanaerobacterium Ascusb_2948 284 VictivallisAscusb_2966 285 Clostridium_IV Ascusb_2983 286 Pelotomaculum Ascusb_2988287 Clostridium_XIVa Ascusb_2990 288 Saccharofermentans Ascusb_3005 289Lachnospiracea_incertae_sedis Ascusb_3008 290 Coprococcus Ascusb_3010291 Clostridium_XIVa Ascusb_3022 292 Clostridium_XIVb Ascusb_3029 293Papillibacter Ascusb_3053 294 Bartonella Ascusb_3056 295 Clostridium_IVAscusb_3058 296 Eubacterium Ascusb_3061 297 Asaccharobacter Ascusb_3066298 Clostridium_IV Ascusb_3073 299 Blautia Ascusb_3074 300 PrevotellaAscusb_3079 301 Ruminococcus Ascusb_3087 302 Selenomonas Ascusb_3120 303Treponema Ascusb_3142 304 Adlercreutzia Ascusb_3147 305 ButyricicoccusAscusb_3161 306 Pseudoflavonifractor Ascusb_3163 307 CorynebacteriumAscusb_3165 308 Adlercreutzia Ascusb_3188 309 Selenomonas Ascusb_3197310 Coraliomargarita Ascusb_3213 311 Paraprevotella Ascusb_3225 312Oscillibacter Ascusb_3229 313 Anaerovorax Ascusb_3240 314Clostridium_XIVa Ascusb_3242 315 Saccharofermentans Ascusb_3248 316Erysipelothrix Ascusb_3263 317 Agaricicola Ascusb_3275 318Denitrobacterium Ascusb_3285 319 Armatimonadetes Ascusb_3299 320Asaccharobacter Ascusb_3304 321 Anaeroplasma Ascusb_3322 322 PrevotellaAscusb_3333 323 Lachnospiracea_incertae_sedis Ascusb_3339 324Clostridium_IV Ascusb_3351 325 Streptococcus Ascusb_3376 326Cellulosilyticum Ascusb_3393 327 Asaccharobacter Ascusb_3405 328Enterorhabdus Ascusb_3408 329 Treponema Ascusb_3415 330 RoseburiaAscusb_3417 331 Victivallis Ascusb_3422 332 Prevotella Ascusb_3424 333Roseburia Ascusb_3446 334 Ruminococcus Ascusb_3451 335 MogibacteriumAscusb_3456 336 Lachnospiracea_incertae_sedis Ascusb_3467 337 PrevotellaAscusb_3479 338 Clostridium_sensu_stricto Ascusb_3480 339 VictivallisAscusb_3481 340 Cyanobacteria Ascusb_3482 341 Treponema Ascusb_3483 342Stenotrophomonas Ascusb_3484 343 Ascusb_3492 344 Clostridium_XIVaAscusb_3494 345 Sphingobium Ascusb_3495 346Lachnospiracea_incertae_sedis Ascusb_3512 347 Oscillibacter Ascusb_3518348 Methylobacterium Ascusb_3523 349 Zhangella Ascusb_3530 350Lachnospiracea_incertae_sedis Ascusb_3545 351 Oscillibacter Ascusb_3546352 Clostridium_III Ascusb_3548 353 Coraliomargarita Ascusb_3563 354Eubacterium Ascusb_3575 355 Enterorhabdus Ascusb_3578 356Clostridium_XIVa Ascusb_3587 357 Saccharofermentans Ascusb_3592 358Clostridium_IV Ascusb_3600 359 Clostridium_sensu_stricto Ascusb_3602 360Victivallis Ascusb_3638 361 Coprococcus Ascusb_3642 362Pseudoflavonifractor Ascusb_3647 363 Anaeroplasma Ascusb_3674 364Anaeroplasma Ascusb_3687 365 Bacteroides Ascusb_3700 366 AcinetobacterAscusb_3717 367 Victivallis Ascusb_3724 368 Victivallis Ascusb_3725 369Mogibacterium Ascusb_3728 370 Oscillibacter Ascusb_3746 371Butyricimonas Ascusb_3748 372 Dethiosulfovibrio Ascusb_3750 373Pseudoflavonifractor Ascusb_3751 374 Clostridium_IV Ascusb_3762 375Anaeroplasma Ascusb_3763 376 Oscillibacter Ascusb_3768 377 HerbiconiuxAscusb_3775 378 Eubacterium Ascusb_3779 379 Armatimonadetes Ascusb_3789380 Selenomonas Ascusb_3796 381 Clostridium_IV Ascusb_3811 382Mogibacterium Ascusb_3825 383 Clostridium_IV Ascusb_3838 384 RoseburiaAscusb_3849 385 Anaerovibrio Ascusb_3866 386 Clostridium_III Ascusb_3875387 Saccharofermentans Ascusb_3903 388 Saccharofermentans Ascusb_3911389 Prevotella Ascusb_3914 390 Clostridium_XIVa Ascusb_3919 391Robinsoniella Ascusb_3950 392 Brevundimonas Ascusb_3952 393Anaerotruncus Ascusb_3970 394 Victivallis Ascusb_3982 395 BacteroidesAscusb_4008 396 Clostridium_XIVb Ascusb_4019 397 Prevotella Ascusb_4033398 Ruminococcus Ascusb_4034 399 Pelobacter Ascusb_4040 400Clostridium_XIVa Ascusb_4063 401 Clostridium_XIVa Ascusb_4067 402Clostridium_XIVb Ascusb_4083 403 Coprococcus Ascusb_4085 404Clostridium_IV Ascusb_4086 405 Clostridium_IV Ascusb_4095 406Coprococcus Ascusb_4114 407 Victivallis Ascusb_4115 408 Clostridium_IIIAscusb_4118 409 Anaerovibrio Ascusb_4120 410 Anaerovorax Ascusb_4124 411Proteiniclasticum Ascusb_4142 412 Anaerovorax Ascusb_4143 413Selenomonas Ascusb_4149 414 Hydrogenoanaerobacterium Ascusb_4155 415Acetanaerobacterium Ascusb_4156 416 Clostridium_XIVa Ascusb_4159 417Asaccharobacter Ascusb_4161 418 Clostridium_XIVa Ascusb_4167 419Lachnospiracea_incertae_sedis Ascusb_4171 420 SaccharofermentansAscusb_4172 421 Prevotella Ascusb_4176 422 Anaeroplasma Ascusb_4179 423Spirochaeta Ascusb_4188 424 Alkaliphilus Ascusb_4213 425 ParaprevotellaAscusb_4215 426 Hippea Ascusb_4217 427 Prevotella Ascusb_4223 428Prevotella Ascusb_4237 429 Hydrogenoanaerobacterium Ascusb_4241 430Clostridium_sensu_stricto Ascusb_4265 431 Paraeggerthella Ascusb_4266432 Clostridium_XIVa Ascusb_4277 433 Clostridium_XIVa Ascusb_4279 434Clostridium_IV Ascusb_4281 435 Clostridium_XIVa Ascusb_4292 436Adhaeribacter Ascusb_4313 437 Syntrophococcus Ascusb_4316 438Clostridium_sensu_stricto Ascusb_4317 439 Saccharofermentans Ascusb_4326440 Clostridium_IV Ascusb_4332 441 Clostridium_IV Ascusb_4345 442Clostridium_sensu_stricto Ascusb_4347 443 Coraliomargarita Ascusb_4375444 Sharpea Ascusb_4380 445 Clostridium_IV Ascusb_4394 446 AnaerovoraxAscusb_4416 447 Blautia Ascusb_4421 448 Clostridium_XIVa Ascusb_4422 449Clostridium_IV Ascusb_4432 450 Anaerovorax Ascusb_4433 451Coraliomargarita Ascusb_4434 452 Lachnospiracea_incertae_sedisAscusb_4442 453 Aquiflexum Ascusb_4449 454 Pedobacter Ascusb_4450 455Robinsoniella Ascusb_4457 456 Pelomonas Ascusb_4468 457Saccharofermentans Ascusb_4469 458 Paracoccus Ascusb_4479 459Enterorhabdus Ascusb_4486 460 Beijerinckia Ascusb_4496 461 SporobacterAscusb_4505 462 Clostridium_IV Ascusb_4517 463 Bacillus Ascusb_4522 464Saccharofermentans Ascusb_4537 465 Spirochaeta Ascusb_4545 466Prevotella Ascusb_4548 467 Eubacterium Ascusb_4556 468 HerbiconiuxAscusb_4559 469 Brevundimonas Ascusb_4560 470 Mogibacterium Ascusb_4563471 Anaerorhabdus Ascusb_4566 472 Victivallis Ascusb_4569 473 PrevotellaAscusb_4573 474 Anaerovorax Ascusb_4579 475 Aquiflexum Ascusb_4606 476Oscillibacter Ascusb_4618 477 Altererythrobacter Ascusb_4626 478Hydrogenoanaerobacterium Ascusb_4627 479 Clostridium_III Ascusb_4634 480Clostridium_XIVb Ascusb_4639 481 Saccharofermentans Ascusb_4644 482Roseburia Ascusb_4652 483 Anaeroplasma Ascusb_4657 484 PlanctomycesAscusb_4676 485 Ruminococcus Ascusb_4679 486 Selenomonas Ascusb_4695 487Anaeroplasma Ascusb_4696 488 Anaerovorax Ascusb_4700 489Rummeliibacillus Ascusb_4701 490 Clostridium_XIVa Ascusb_4716 491Anaeroplasma Ascusb_4731 492 Butyrivibrio Ascusb_4737 493Lachnospiracea_incertae_sedis Ascusb_4738 494 Anaerotruncus Ascusb_4758495 Syntrophococcus Ascusb_4763 496 Paraeggerthella Ascusb_4795 497Papillibacter Ascusb_4800 498 Lachnospiracea_incertae_sedis Ascusb_4805499 Prevotella Ascusb_4820 500 Papillibacter Ascusb_4828 501Streptococcus Ascusb_4852 502 Methanobrevibacter Ascusb_4859 503Prevotella Ascusb_4861 504 Prevotella Ascusb_4867 505 PrevotellaAscusb_4873 506 Coraliomargarita Ascusb_4882 507 Prevotella Ascusb_4886508 Thermotalea Ascusb_4893 509 Clostridium_XIVa Ascusb_4897 510Atopobium Ascusb_4945 511 Prevotella Ascusb_4969 512 MogibacteriumAscusb_4972 513 Clostridium_XIVa Ascusb_4976 514 Clostridium_XIVaAscusb_4997 515 Eggerthella Ascusb_4999 516 Blautia Ascusb_5000 517Vampirovibrio Ascusb_5006 518 Papillibacter Ascusb_5040 519 BeijerinckiaAscusb_5058 520 Bacteroides Ascusb_5060 521 Desulfotomaculum Ascusb_5065522 Acidobacteria Ascusb_5069 523 Clostridium_XIVa Ascusb_5081 524Clostridium_XIVa Ascusb_5089 525 Clostridium_XIVa Ascusb_5095 526Cryptanaerobacter Ascusb_5103 527 Prevotella Ascusb_5113 528Syntrophomonas Ascusb_5137 529 Erysipelothrix Ascusb_5144 530Selenomonas Ascusb_5165 531 Clostridium_III Ascusb_5171 532Flavobacterium Ascusb_5181 533 Thermotalea Ascusb_5191 534Lachnospiracea_incertae_sedis Ascusb_5194 535 MucilaginibacterAscusb_5197 536 Bacteroides Ascusb_5198 537 Ruminococcus Ascusb_5206 538Clostridium_XIVa Ascusb_5223 539 Asaccharobacter Ascusb_5225 540 BlautiaAscusb_5235 541 Mucilaginibacter Ascusb_5247 542 Coprococcus Ascusb_5252543 Lachnospiracea_incertae_sedis Ascusb_5253 544 ButyricimonasAscusb_5255 545 Lachnospiracea_incertae_sedis Ascusb_5267 546 TreponemaAscusb_5280 547 Clostridium_sensu_stricto Ascusb_5281 548Clostridium_XIVa Ascusb_5289 549 Anaerovorax Ascusb_5292 550Saccharofermentans Ascusb_5294 551 Clostridium_XIVa Ascusb_5295 552Clostridium_III Ascusb_5301 553 Clostridium_IV Ascusb_5313 554Ruminococcus Ascusb_5324 555 Clostridium_XIVa Ascusb_5326 556Clostridium_XI Ascusb_5335 557 Clostridium_XIVa Ascusb_5336 558Eubacterium Ascusb_5338 559 Lachnospiracea_incertae_sedis Ascusb_5342560 Clostridium_IV Ascusb_5352 561 Ruminococcus Ascusb_5353 562Clostridium_IV Ascusb_5354 563 Faecalibacterium Ascusb_5360 564Anaerovibrio Ascusb_5368 565 Asaccharobacter Ascusb_5397 566Pelotomaculum Ascusb_5411 567 Spirochaeta Ascusb_5422 568 PrevotellaAscusb_5429 569 Lachnospiracea_incertae_sedis Ascusb_5440 570Anaerovorax Ascusb_5441 571 Clostridium_IV Ascusb_5443 572 VictivallisAscusb_5451 573 Syntrophococcus Ascusb_5456 574 SyntrophococcusAscusb_5463 575 Desulfovibrio Ascusb_5481 576Lachnospiracea_incertae_sedis Ascusb_5485 577Lachnospiracea_incertae_sedis Ascusb_5495 578 Clostridium_IV Ascusb_5509579 Prevotella Ascusb_5510 580 Victivallis Ascusb_5512 581Clostridium_XIVa Ascusb_5515 582 Selenomonas Ascusb_5517 583 BacteroidesAscusb_5530 584 Clostridium_XIVa Ascusb_5536 585 Eggerthella Ascusb_5554586 Selenomonas Ascusb_5584 587 Mogibacterium Ascusb_5592 588Armatimonadetes Ascusb_5609 589 Clostridium_XIVa Ascusb_5612 590Victivallis Ascusb_5623 591 Paraprevotella Ascusb_5628 592 BrevundimonasAscusb_5647 593 Prevotella Ascusb_5650 594 Prevotella Ascusb_5652 595Robinsoniella Ascusb_5660 596 Clostridium_III Ascusb_5686 597Butyricimonas Ascusb_5689 598 Spirochaeta Ascusb_5691 599Hydrogenoanaerobacterium Ascusb_5694 600 Proteiniclasticum Ascusb_5716601 Roseburia Ascusb_5725 602 Clostridium_XIVa Ascusb_5738 603Anaerofustis Ascusb_5746 604 Succiniclasticum Ascusb_5765 605Anaeroplasma Ascusb_5770 606 Oscillibacter Ascusb_5777 607Escherichia/Shigella Ascusb_5789 608 Bacteroides Ascusb_5812 609Clostridium_XIVa Ascusb_5830 610 Clostridium_XIVa Ascusb_5838 611Clostridium_IV Ascusb_5841 612 Clostridium_III Ascusb_5845 613Prevotella Ascusb_5847 614 Coprococcus Ascusb_5849 615 OscillibacterAscusb_5858 616 Parabacteroides Ascusb_5862 617 Bacteroides Ascusb_5868618 Mogibacterium Ascusb_5869 619 Solobacterium Ascusb_5870 620Bacteroides Ascusb_5874 621 Clostridium_III Ascusb_5877 622 VictivallisAscusb_5879 623 Saccharofermentans Ascusb_5884 624 SaccharofermentansAscusb_5889 625 Olivibacter Ascusb_5894 626 Thermotalea Ascusb_5895 627Proteiniclasticum Ascusb_5913 628 Clostridium_III Ascusb_5926 629Anaeroplasma Ascusb_5934 630 Treponema Ascusb_5939 631 Clostridium_XIVaAscusb_5940 632 Clostridium_III Ascusb_5950 633 DesulfotomaculumAscusb_5953 634 Bacillus Ascusb_5969 635 Anaerovorax Ascusb_5972 636Ruminococcus Ascusb_5973 637 Agarivorans Ascusb_5975 638 AnaerotruncusAscusb_5979 639 Papillibacter Ascusb_5984 640 Clostridium_XIVaAscusb_5991 641 Clostridium_III Ascusb_5996 642 Bacteroides Ascusb_5997643 Clostridium_XIVa Ascusb_5998 644 Ruminococcus Ascusb_6003 645Clostridium_XIVa Ascusb_6005 646 Oscillibacter Ascusb_6006 647Nitrobacter Ascusb_6022 648 Clostridium_XIVa Ascusb_6026 649Lachnospiracea_incertae_sedis Ascusb_6035 650 Limibacter Ascusb_6037 651Desulfovibrio Ascusb_6053 652 Coprococcus Ascusb_6067 653 AnaerovoraxAscusb_6070 654 Spirochaeta Ascusb_6074 655 Cyanobacteria Ascusb_6079656 Saccharofermentans Ascusb_6081 657 Anaeroplasma Ascusb_6106 658Clostridium_III Ascusb_6115 659 Victivallis Ascusb_6151 660Enterorhabdus Ascusb_6168 661 Clostridium_IV Ascusb_6169 662Erysipelothrix Ascusb_6172 663 Clostridium_III Ascusb_6200 664Clostridium_sensu_stricto Ascusb_6207 665 Gelidibacter Ascusb_6212 666Roseburia Ascusb_6219 667 Neisseria Ascusb_6270 668 PrevotellaAscusb_6273 669 Cyanobacteria Ascusb_6275 670 Oscillibacter Ascusb_6282671 Candidate phylum TM7 Ascusb_6313 672 Prevotella Ascusb_6326 673Saccharofermentans Ascusb_6330 674 Erysipelotrichaceae_incertae_sedisAscusb_6337 675 Spirochaeta Ascusb_6342 676 Clostridium_XIVa Ascusb_6372677 Clostridium_XIVb Ascusb_6376 678 Clostridium_XIVa Ascusb_6387 679Adlercreutzia Ascusb_6389 680 Clostridium_XIVa Ascusb_6394 681Lachnospiracea_incertae_sedis Ascusb_6400 682 Clostridium_IV Ascusb_6403683 Adlercreutzia Ascusb_6406 684 Prevotella Ascusb_6409 685Syntrophococcus Ascusb_6420 686 Treponema Ascusb_6433 687 PrevotellaAscusb_6448 688 Clostridium_III Ascusb_6450 689 PseudoflavonifractorAscusb_6463 690 Clostridium_IV Ascusb_6468 691 Sharpea Ascusb_6473 692Dongia Ascusb_6499 693 Eubacterium Ascusb_6505 694 PrevotellaAscusb_6507 695 Clostridium_IV Ascusb_6519 696 ParabacteroidesAscusb_6525 697 Brevundimonas Ascusb_6535 698 Clostridium_XIVaAscusb_6540 699 Ruminococcus Ascusb_6541 700 Thermotalea Ascusb_6558 701Victivallis Ascusb_6561 702 Anaeroplasma Ascusb_6563 703 OscillibacterAscusb_6564 704 Ruminococcus Ascusb_6570 705 Clostridium_XIVaAscusb_6578 706 Clostridium_XIVa Ascusb_6581 707 Clostridium_IVAscusb_6586 708 Roseburia Ascusb_6593 709 Eggerthella Ascusb_6612 710Clostridium_III Ascusb_6614 711 Clostridium_XIVa Ascusb_6621 712Lactobacillus Ascusb_6630 713 Bacteroides Ascusb_6633 714Cellulosilyticum Ascusb_6635 715 Brevundimonas Ascusb_6645 716Clostridium_IV Ascusb_6670 717 Prevotella Ascusb_6672 718 HelicobacterAscusb_6676 719 Clostridium_IV Ascusb_6683 720 ProteiniclasticumAscusb_6684 721 Brevundimonas Ascusb_6701 722 Clostridium_XIVaAscusb_6704 723 Prevotella Ascusb_6706 724 Desulfovibrio Ascusb_6708 725Coraliomargarita Ascusb_6709 726 Eubacterium Ascusb_6715 727Sphingomonas Ascusb_6718 728 Prevotella Ascusb_6730 729 Clostridium_IVAscusb_6734 730 Paraprevotella Ascusb_6735 731 Ruminococcus Ascusb_6746732 Saccharofermentans Ascusb_6756 733 Clostridium_III Ascusb_6757 734Clostridium_III Ascusb_6774 735 Turicibacter Ascusb_6792 736 PrevotellaAscusb_6796 737 Clostridium_XIVa Ascusb_6803 738 Fusibacter Ascusb_6813739 Clostridium_XIVa Ascusb_6824 740 Clostridium_IV Ascusb_6833 741Rummeliibacillus Ascusb_6848 742 Mogibacterium Ascusb_6852 743Bacteroides Ascusb_6864 744 Pelospora Ascusb_6875 745 EggerthellaAscusb_6880 746 Eubacterium Ascusb_6887 747 Blautia Ascusb_6889 748Clostridium_XIVb Ascusb_6901 749 Ehrlichia Ascusb_6907 750 EubacteriumAscusb_6930 751 Prevotella Ascusb_6943 752 Clostridium_XIVa Ascusb_6952753 Treponema Ascusb_6954 754 Hydrogenoanaerobacterium Ascusb_6957 755Selenomonas Ascusb_6964 756 Saccharofermentans Ascusb_6966 757Clostridium_IV Ascusb_6971 758 Clostridium_sensu_stricto Ascusb_6976 759Anaerovorax Ascusb_6979 760 Spirochaeta Ascusb_6997 761 BrevundimonasAscusb_7001 762 Eubacterium Ascusb_7017 763 Clostridium_XIVa Ascusb_7025764 Anaerovorax Ascusb_7031 765 Ruminococcus Ascusb_7039 766Papillibacter Ascusb_7040 767 Clostridium_IV Ascusb_7043 768Hydrogenoanaerobacterium Ascusb_7046 769 Asaccharobacter Ascusb_7048 770Clostridium_XIVa Ascusb_7054 771 Rhodocista Ascusb_7078 772Clostridium_XIVa Ascusb_7087 773 Beijerinckia Ascusb_7091 774Lactobacillus Ascusb_7101 775 Cryptanaerobacter Ascusb_7102 776Prevotella Ascusb_7113 777 Anaerovibrio Ascusb_7114 778 AnaerovoraxAscusb_7123 779 Lachnospiracea_incertae_sedis Ascusb_7128 780Enterorhabdus Ascusb_7131 781 Clostridium_XIVb Ascusb_7141 782Selenomonas Ascusb_7148 783 Eubacterium Ascusb_7149 784 ThermotaleaAscusb_7151 785 Enterorhabdus Ascusb_7153 786 Clostridium_IIIAscusb_7159 787 Acetanaerobacterium Ascusb_7164 788 TreponemaAscusb_7168 789 Clostridium_XIVa Ascusb_7176 790 EnterorhabdusAscusb_7180 791 Prevotella Ascusb_7188 792 Desulfovibrio Ascusb_7199 793Aminobacter Ascusb_7213 794 Clostridium_IV Ascusb_7224 795 RikenellaAscusb_7225 796 Gordonibacter Ascusb_7240 797 Papillibacter Ascusb_7245798 Syntrophococcus Ascusb_7246 799 Clostridium_sensu_strictoAscusb_7256 800 Hahella Ascusb_7257 801 Vampirovibrio Ascusb_7264 802Coprococcus Ascusb_7275 803 Coraliomargarita Ascusb_7299 804Clostridium_III Ascusb_7300 805 Clostridium_XIVa Ascusb_7304 806Desulfotomaculum Ascusb_7325 807 Helicobacter Ascusb_7373 808Syntrophococcus Ascusb_7380 809 Lachnospiracea_incertae_sedisAscusb_7384 810 Clostridium_IV Ascusb_7385 811 Paludibacter Ascusb_7395812 Lachnospiracea_incertae_sedis Ascusb_7401 813Lachnospiracea_incertae_sedis Ascusb_7412 814 Adhaeribacter Ascusb_7419815 Clostridium_IV Ascusb_7420 816 Cryptanaerobacter Ascusb_7424 817Idiomarina Ascusb_7435 818 Clostridium_IV Ascusb_7437 819 SelenomonasAscusb_7440 820 Acetanaerobacterium Ascusb_7444 821 BifidobacteriumAscusb_7446 822 Clostridium_XIVb Ascusb_7449 823 AsaccharobacterAscusb_7450 824 Eubacterium Ascusb_7452 825 Anaeroplasma Ascusb_7455 826Saccharofermentans Ascusb_7456 827 Ruminococcus Ascusb_7467 828Clostridium_III Ascusb_7470 829 Acholeplasma Ascusb_7472 830 PedobacterAscusb_7476 831 Sphingomonas Ascusb_7487 832 Verrucomicrobia Ascusb_7525833 Anaerovorax Ascusb_7533 834 Spirochaeta Ascusb_7534 835Paraeggerthella Ascusb_7539 836 Lachnospiracea_incertae_sedisAscusb_7542 837 Bacteroides Ascusb_7543 838 Paenibacillus Ascusb_7549839 Prevotella Ascusb_7553 840 Bacteroides Ascusb_7555 841Clostridium_XIVa Ascusb_7563 842 Clostridium_XIVa Ascusb_7568 843Roseburia Ascusb_7572 844 Clostridium_XIVa Ascusb_7581 845Clostridium_III Ascusb_7591 846 Pedobacter Ascusb_7599 847 RobinsoniellaAscusb_7614 848 Anaeroplasma Ascusb_7615 849 Clostridium_XIVaAscusb_7622 850 Hydrogenoanaerobacterium Ascusb_7626 851 TuricibacterAscusb_7638 852 Papillibacter Ascusb_7645 853 Clostridium_XIVaAscusb_7647 854 Saccharofermentans Ascusb_7648 855 Clostridium_XIVbAscusb_7650 856 Sporobacter Ascusb_7662 857 Asaccharobacter Ascusb_7663858 Bacteroides Ascusb_7669 859 Anaeroplasma Ascusb_7677 860 SporobacterAscusb_7680 861 Streptomyces Ascusb_7690 862 Arcobacter Ascusb_7694 863Clostridium_XIVa Ascusb_7699 864 Barnesiella Ascusb_7706 865Lactobacillus Ascusb_7723 866 Flavobacterium Ascusb_7728 867 VictivallisAscusb_7733 868 Clostridium_XIVa Ascusb_7735 869 Ureaplasma Ascusb_7748870 Acetanaerobacterium Ascusb_7752 871 Slackia Ascusb_7753 872Lachnospiracea_incertae_sedis Ascusb_7761 873 Oscillibacter Ascusb_7763874 Prevotella Ascusb_7765 875 Proteiniphilum Ascusb_7767 876Spirochaeta Ascusb_7784 877 Ruminococcus Ascusb_7788 878 PrevotellaAscusb_7792 879 Butyricicoccus Ascusb_7796 880 Devosia Ascusb_7817 881Anaeroplasma Ascusb_7828 882 Oscillibacter Ascusb_7829 883 BarnesiellaAscusb_7831 884 Atopobium Ascusb_7837 885 Clostridium_XIVa Ascusb_7838886 Methanobrevibacter Ascusb_7839 887 Butyricimonas Ascusb_7849 888Butyricimonas Ascusb_7853 889 Asaccharobacter Ascusb_7855 890Enhydrobacter Ascusb_7871 891 Treponema Ascusb_7872 892 Clostridium_XIVaAscusb_7873 893 Adlercreutzia Ascusb_7874 894 Prevotella Ascusb_7890 895Pseudoflavonifractor Ascusb_7896 896 Syntrophococcus Ascusb_7898 897Clostridium_IV Ascusb_7901 898 Demequina Ascusb_7902 899Lachnospiracea_incertae_sedis Ascusb_7904 900 SaccharofermentansAscusb_7924 901 Sphaerisporangium Ascusb_7925 902 AnaeroplasmaAscusb_7939 903 Geobacillus Ascusb_7958 904 Prevotella Ascusb_7959 905Clostridium_XIVa Ascusb_7967 906 Victivallis Ascusb_7973 907 BacteroidesAscusb_7989 908 Demequina Ascusb_7990 909 Paraeggerthella Ascusb_7994910 Paraprevotella Ascusb_7996 911 Pseudoflavonifractor Ascusb_8013 912Roseburia Ascusb_8018 913 Gelidibacter Ascusb_8038 914 Clostridium_IVAscusb_8069 915 Rhizobium Ascusb_8076 916 Acholeplasma Ascusb_8081 917Clostridium_XIVa Ascusb_8084 918 Bacteroides Ascusb_8091 919 BacteroidesAscusb_8105 920 Papillibacter Ascusb_8107 921 Fusibacter Ascusb_8113 922Coraliomargarita Ascusb_8120 923 Papillibacter Ascusb_8123 924Clostridium_XIVa Ascusb_8149 925 Acholeplasma Ascusb_8167 926Catenibacterium Ascusb_8169 927 Clostridium_IV Ascusb_8172 928Clostridium_IV Ascusb_8173 929 Clostridium_IV Ascusb_8179 930Nitrobacter Ascusb_8182 931 Victivallis Ascusb_8189 932 SelenomonasAscusb_8196 933 Enterorhabdus Ascusb_8200 934 Eubacterium Ascusb_8202935 Roseburia Ascusb_8206 936 Prevotella Ascusb_8211 937 AsaccharobacterAscusb_8222 938 Bacteroides Ascusb_8230 939 Clostridium_XIVa Ascusb_8238940 Gelidibacter Ascusb_8245 941 Brevundimonas Ascusb_8254 942Clostridium_XIVa Ascusb_8260 943 Prevotella Ascusb_8266 944Oscillibacter Ascusb_8268 945 Asteroleplasma Ascusb_8280 946Anaeroplasma Ascusb_8283 947 Oscillibacter Ascusb_8311 948 BilophilaAscusb_8317 949 Oscillibacter Ascusb_8318 950 Clostridium_IV Ascusb_8320951 Prevotella Ascusb_8321 952 Geosporobacter Ascusb_8329 953Butyricimonas Ascusb_8363 954 Pseudoflavonifractor Ascusb_8366 955Barnesiella Ascusb_8367 956 Selenomonas Ascusb_8370 957 PrevotellaAscusb_8374 958 Enterorhabdus Ascusb_8379 959 Oscillibacter Ascusb_8384960 Pelotomaculum Ascusb_8394 961 Cellulosilyticum Ascusb_8396 962Clostridium_IV Ascusb_8402 963 Parabacteroides Ascusb_8410 964Papillibacter Ascusb_8413 965 Bacteroides Ascusb_8439 966 PrevotellaAscusb_8440 967 Hydrogenoanaerobacterium Ascusb_8447 968Clostridium_XIVa Ascusb_8470 969 Prevotella Ascusb_8480 970Clostridium_IV Ascusb_8484 971 Howardella Ascusb_8487 972 SlackiaAscusb_8498 973 Methylobacter Ascusb_8500 974 Treponema Ascusb_8508 975Clostridium_XIVa Ascusb_8514 976 Devosia Ascusb_8518 977 RuminococcusAscusb_8537 978 Lachnospiracea_incertae_sedis Ascusb_8569 979Clostridium_III Ascusb_8580 980 Methanobrevibacter Ascusb_8595 981Paraprevotella Ascusb_8600 982 Desulfobulbus Ascusb_8627 983Butyricicoccus Ascusb_8639 984 Clostridium_XIVa Ascusb_8657 985Dialister Ascusb_8669 986 Selenomonas Ascusb_8681 987 SpirochaetaAscusb_8696 988 Clostridium_IV Ascusb_8712 989 CellulosilyticumAscusb_8713 990 Prevotella Ascusb_8714 991 PseudoflavonifractorAscusb_8715 992 Clostridium_III Ascusb_8728 993 OscillibacterAscusb_8733 994 Faecalibacterium Ascusb_8746 995 Clostridium_XIVbAscusb_8753 996 Eubacterium Ascusb_8758 997 Clostridium_III Ascusb_8762998 Prevotella Ascusb_8769 999 Paenibacillus Ascusb_8771 1000 PedobacterAscusb_8782 1001 Butyricicoccus Ascusb_8786 1002 Clostridium_XIVaAscusb_8787 1003 Roseburia Ascusb_8799 1004 HydrogenoanaerobacteriumAscusb_8804 1005 Adhaeribacter Ascusb_8807 1006 Eubacterium Ascusb_88151007 Bacteroides Ascusb_8822 1008 Victivallis Ascusb_8835 1009 RoseburiaAscusb_8840 1010 Treponema Ascusb_8857 1011 Prevotella Ascusb_8860 1012Prevotella Ascusb_8870 1013 Hydrogenoanaerobacterium Ascusb_8873 1014Clostridium_XIVa Ascusb_8883 1015 Bacteroides Ascusb_8884 1016Bacteroides Ascusb_8886 1017 Lactobacillus Ascusb_8888 1018Adlercreutzia Ascusb_8892 1019 Dethiosulfovibrio Ascusb_8916 1020Lutispora Ascusb_8934 1021 Turicibacter Ascusb_8942 1022 CyanobacteriaAscusb_8953 1023 Clostridium_sensu_stricto Ascusb_8956 1024Cyanobacteria Ascusb_8972 1025 Bulleidia Ascusb_9004 1026 AquiflexumAscusb_9015 1027 Lachnospiracea_incertae_sedis Ascusb_9026 1028Lachnospiracea_incertae_sedis Ascusb_9073 1029 Clostridium_IIIAscusb_9075 1030 Roseburia Ascusb_9081 1031 Glaciecola Ascusb_9086 1032Clostridium_XIVa Ascusb_9090 1033 Hydrogenoanaerobacterium Ascusb_90951034 Clostridium_IV Ascusb_9097 1035 Sphaerobacter Ascusb_9098 1036Cyanobacteria Ascusb_9105 1037 Prevotella Ascusb_9109 1038 TuricibacterAscusb_9112 1039 Ruminococcus Ascusb_9122 1040 Clostridium_IVAscusb_9131 1041 Clostridium_XIVa Ascusb_9145 1042 SaccharofermentansAscusb_9151 1043 Clostridium_XIVb Ascusb_9154 1044 RuminococcusAscusb_9160 1045 Fibrobacter Ascusb_9169 1046 ProteiniclasticumAscusb_9176 1047 Anaeroplasma Ascusb_9178 1048 Cyanobacteria Ascusb_91841049 Algoriphagus Ascusb_9189 1050 Clostridium_XIVa Ascusb_9196 1051Howardella Ascusb_9200 1052 Clostridium_XIVa Ascusb_9201 1053Barnesiella Ascusb_9211 1054 Clostridium_IV Ascusb_9234 1055 PrevotellaAscusb_9238 1056 Clostridium_XIVa Ascusb_9251 1057 ButyricimonasAscusb_9261 1058 Blautia Ascusb_9264 1059 Prevotella Ascusb_9274 1060Clostridium_XIVa Ascusb_9277 1061 Blautia Ascusb_9282 1062Clostridium_IV Ascusb_9291 1063 Flavobacterium Ascusb_9292 1064Prevotella Ascusb_9300 1065 Clostridium_XIVa Ascusb_9301 1066Clostridium_XIVa Ascusb_9302 1067 Eubacterium Ascusb_9313 1068Butyricicoccus Ascusb_9340 1069 Fluviicola Ascusb_9343 1070 AnaerovibrioAscusb_9354 1071 Blautia Ascusb_9355 1072 Verrucomicrobia Ascusb_93671073 Clostridium_sensu_stricto Ascusb_9368 1074 Spirochaeta Ascusb_93691075 Clostridium_XI Ascusb_9372 1076 Anaerovorax Ascusb_9376 1077Roseburia Ascusb_9381 1078 Mucilaginibacter Ascusb_9388 1079Clostridium_XI Ascusb_9389 1080 Lachnospiracea_incertae_sedisAscusb_9401 1081 Prevotella Ascusb_9402 1082 Clostridium_III Ascusb_94111083 Lachnospiracea_incertae_sedis Ascusb_9415 1084 CoprococcusAscusb_9427 1085 Acholeplasma Ascusb_9432 1086 Clostridium_IIIAscusb_9453 1087 Lactobacillus Ascusb_9454 1088 Clostridium_IVAscusb_9455 1089 Prevotella Ascusb_9465 1090 Bifidobacterium Ascusb_94971091 Adhaeribacter Ascusb_9507 1092 Hydrogenoanaerobacterium Ascusb_95181093 Acetivibrio Ascusb_9521 1094 Cyanobacteria Ascusb_9532 1095Flammeovirga Ascusb_9535 1096 Dethiosulfovibrio Ascusb_9543 1097 HippeaAscusb_9545 1098 Faecalibacterium Ascusb_9558 1099 SpirochaetaAscusb_9559 1100 Brevundimonas Ascusb_9563 1101 MucilaginibacterAscusb_9564 1102 Hydrogenoanaerobacterium Ascusb_9580 1103Asaccharobacter Ascusb_9587 1104 Clostridium_IV Ascusb_9591 1105Mogibacterium Ascusb_9605 1106 Clostridium_IV Ascusb_9617 1107Oscillibacter Ascusb_9619 1108 Clostridium_XIVa Ascusb_9628 1109Faecalibacterium Ascusb_9640 1110 Altererythrobacter Ascusb_9644 1111Gelidibacter Ascusb_9656 1112 Prevotella Ascusb_9662 1113 AnaerovoraxAscusb_9663 1114 Riemerella Ascusb_9664 1115 SphingobacteriumAscusb_9666 1116 Syntrophococcus Ascusb_9668 1117 BacteroidesAscusb_9669 1118 Papillibacter Ascusb_9678 1119 ButyricicoccusAscusb_9679 1120 Clostridium_IV Ascusb_9680 1121Hydrogenoanaerobacterium Ascusb_9684 1122 Marvinbryantia Ascusb_96881123 Brevibacillus Ascusb_9701 1124 Clostridium_IV Ascusb_9715 1125Prevotella Ascusb_9719 1126 Clostridium_IV Ascusb_9734 1127 AminobacterAscusb_9759 1128 Sporotomaculum Ascusb_9764 1129 Clostridium_IVAscusb_9779 1130 Pedobacter Ascusb_9780 1131 Victivallis Ascusb_97821132 Gelidibacter Ascusb_9792 1133 Prevotella Ascusb_9824 1134Wautersiella Ascusb_9839 1135 Slackia Ascusb_9846 1136 PyramidobacterAscusb_9851 1137 Lachnospiracea_incertae_sedis Ascusb_9862 1138Clostridium_XIVa Ascusb_9869 1139 Prevotella Ascusb_9876 1140Lentisphaera Ascusb_9886 1141 Desulfoluna Ascusb_9895 1142Clostridium_III Ascusb_9897 1143 Clostridium_sensu_stricto Ascusb_99251144 Prevotella Ascusb_9929 1145 Clostridium_III Ascusb_9934 1146Clostridium_IV Ascusb_9949 1147 Prevotella Ascusb_9951 1148Cyanobacteria Ascusb_9954 1149 Helicobacter Ascusb_9958 1150Clostridium_XIVa Ascusb_9977 1151 Coprococcus Ascusb_9982 1152Bradyrhizobium Ascusb_9993 1153 Clostridium_IV Ascusb_9996 1154Sphingobacterium Ascusb_10002 1155 Gelidibacter Ascusb_10023 1156Vasilyevaea Ascusb_10029 1157 Eubacterium Ascusb_10030 1158Clostridium_XIVa Ascusb_10034 1159 Eubacterium Ascusb_10044 1160Syntrophococcus Ascusb_10045 1161 Prevotella Ascusb_10050 1162 TreponemaAscusb_10057 1163 Anaerovorax Ascusb_10058 1164Erysipelotrichaceae_incertae_sedis Ascusb_10059 1165 SulfurovumAscusb_10084 1166 Clostridium_IV Ascusb_10085 1167 PapillibacterAscusb_10087 1168 Paracoccus Ascusb_10094 1169 HydrogenoanaerobacteriumAscusb_10102 1170 Adhaeribacter Ascusb_10121 1171Lachnospiracea_incertae_sedis Ascusb_10126 1172 Bacteroides Ascusb_101271173 Hydrogenoanaerobacterium Ascusb_10129 1174 TelmatospirillumAscusb_10138 1175 Clostridium_XIVa Ascusb_10144 1176Hydrogenoanaerobacterium Ascusb_10147 1177 Clostridium_IV Ascusb_101561178 Vasilyevaea Ascusb_10164 1179 Anaeroplasma Ascusb_10177 1180Sporotomaculum Ascusb_10193 1181 Clostridium_IV Ascusb_10194 1182Enterorhabdus Ascusb_10204 1183 Bacteroides Ascusb_10208 1184Anaerotruncus Ascusb_10210 1185 Rhodopirellula Ascusb_10215 1186Clostridium_XIVa Ascusb_10221 1187 Gelidibacter Ascusb_10243 1188Anaerofustis Ascusb_10268 1189 Butyricicoccus Ascusb_10269 1190Butyricicoccus Ascusb_10278 1191 Clostridium_XIVa Ascusb_10281 1192Cryptanaerobacter Ascusb_10284 1193 Clostridium_XIVa Ascusb_10299 1194Mogibacterium Ascusb_10309 1195 Syntrophococcus Ascusb_10313 1196Bacteroides Ascusb_10325 1197 Treponema Ascusb_10327 1198Coraliomargarita Ascusb_10344 1199 Ruminococcus Ascusb_10368 1200Prevotella Ascusb_10374 1201 Pseudaminobacter Ascusb_10380 1202Prevotella Ascusb_10392 1203 Treponema Ascusb_10450 1204 SyntrophococcusAscusb_10456 1205 Clostridium_IV Ascusb_10457 1206 TenacibaculumAscusb_10462 1207 Parabacteroides Ascusb_10466 1208 LuteimonasAscusb_10469 1209 Eubacterium Ascusb_10488 1210 Roseburia Ascusb_104951211 Oscillibacter Ascusb_10504 1212 Cyanobacteria Ascusb_10529 1213Prevotella Ascusb_10547 1214 Clostridium_IV Ascusb_10548 1215 TreponemaAscusb_10557 1216 Clostridium_IV Ascusb_10561 1217 VictivallisAscusb_10562 1218 Clostridium_XIVa Ascusb_10576 1219 OscillibacterAscusb_10586 1220 Papillibacter Ascusb_10598 1221 CellulosilyticumAscusb_10604 1222 Treponema Ascusb_10607 1223 Ruminococcus Ascusb_106091224 Coraliomargarita Ascusb_10612 1225 Butyricicoccus Ascusb_10613 1226Blautia Ascusb_10615 1227 Lachnospiracea_incertae_sedis Ascusb_106171228 Prevotella Ascusb_10622 1229 Clostridium_IV Ascusb_10623 1230Clostridium_IV Ascusb_10635 1231 Clostridium_III Ascusb_10655 1232Neptunomonas Ascusb_10677 1233 Clostridium_IV Ascusb_10682 1234Howardella Ascusb_10685 1235 Clostridium_IV Ascusb_10687 1236 RoseburiaAscusb_10711 1237 Oscillibacter Ascusb_10739 1238 Clostridium_XIVaAscusb_10740 1239 Clostridium_IV Ascusb_10741 1240 SporobacterAscusb_10749 1241 Clostridium_XIVa Ascusb_10769 1242 ButyricicoccusAscusb_10774 1243 Clostridium_XIVa Ascusb_10787 1244 FilomicrobiumAscusb_10788 1245 Bacteroides Ascusb_10790 1246 Clostridium_XIVaAscusb_10809 1247 Brevundimonas Ascusb_10812 1248 Clostridium_IVAscusb_10817 1249 Paracoccus Ascusb_10818 1250 Schlegelella Ascusb_108371251 Clostridium_XI Ascusb_10844 1252 Diaphorobacter Ascusb_10847 1253Clostridium_sensu_stricto Ascusb_10858 1254 SaccharopolysporaAscusb_10863 1255 Prevotella Ascusb_10871 1256 Eggerthella Ascusb_108781257 Gelidibacter Ascusb_10888 1258 Prevotella Ascusb_10899 1259Pseudomonas Ascusb_10922 1260 Prevotella Ascusb_10927 1261 PrevotellaAscusb_10937 1262 Prevotella Ascusb_10940 1263 BrevundimonasAscusb_10945 1264 Bacteroides Ascusb_10982 1265 Clostridium_XIVaAscusb_11015 1266 Photobacterium Ascusb_11027 1267 Clostridium_XIVaAscusb_11031 1268 Clostridium_XIVb Ascusb_11032 1269 PrevotellaAscusb_11037 1270 Clostridium_IV Ascusb_11046 1271 AnaeroplasmaAscusb_11051 1272 Caldilinea Ascusb_11053 1273 Clostridium_XIVaAscusb_11059 1274 Victivallis Ascusb_11061 1275 BrevundimonasAscusb_11063 1276 Cyanobacteria Ascusb_11074 1277 PrevotellaAscusb_11120 1278 Slackia Ascusb_11124 1279 Pedobacter Ascusb_11125 1280Prevotella Ascusb_11129 1281 Trueperella Ascusb_11141 1282 OscillibacterAscusb_11170 1283 Cyanobacteria Ascusb_11185 1284 VictivallisAscusb_11199 1285 Bacteroides Ascusb_11200 1286 Micrococcus Ascusb_112071287 Olivibacter Ascusb_11209 1288 Anaerophaga Ascusb_11211 1289Selenomonas Ascusb_11214 1290 Megasphaera Ascusb_11219 1291Clostridium_XIVa Ascusb_11221 1292 Clostridium_XIVa Ascusb_11241 1293Eubacterium Ascusb_11245 1294 Cyanobacteria Ascusb_11253 1295Clostridium_XIVa Ascusb_11287 1296 Treponema Ascusb_11288 1297Cryptanaerobacter Ascusb_11289 1298 Xanthomonas Ascusb_11301 1299Asteroleplasma Ascusb_11302 1300 Cyanobacteria Ascusb_11315 1301Sporotomaculum Ascusb_11321 1302 Bacteroides Ascusb_11324 1303Asaccharobacter Ascusb_11330 1304 Clostridium_IV Ascusb_11343 1305Cyanobacteria Ascusb_11348 1306 Clostridium_XIVa Ascusb_11362 1307Treponema Ascusb_11365 1308 Prevotella Ascusb_11384 1309 TuricibacterAscusb_11388 1310 Clostridium_IV Ascusb_11389 1311 Clostridium_IVAscusb_11397 1312 Clostridium_IV Ascusb_11403 1313 OscillibacterAscusb_11410 1314 Deinococcus Ascusb_11423 1315 Pedobacter Ascusb_114271316 Anaerovorax Ascusb_11435 1317 Clostridium_IV Ascusb_11442 1318Bacteroides Ascusb_11445 1319 Clostridium_IV Ascusb_11461 1320Rhodococcus Ascusb_11463 1321 Treponema Ascusb_11464 1322Mucilaginibacter Ascusb_11475 1323 Clostridium_XIVa Ascusb_11503 1324Olivibacter Ascusb_11510 1325 Clostridium_XIVa Ascusb_11519 1326Barnesiella Ascusb_11581 1327 Clostridium_XIVb Ascusb_11584 1328Gelidibacter Ascusb_11600 1329 Methanobrevibacter Ascusb_11602 1330Anaerotruncus Ascusb_11612 1331 Lachnospiracea_incertae_sedisAscusb_11653 1332 Erysipelotrichaceae_incertae_sedis Ascusb_11656 1333Mesorhizobium Ascusb_11681 1334 Clostridium_XI Ascusb_11695 1335Planctomyces Ascusb_11698 1336 Aerococcus Ascusb_11713 1337 VictivallisAscusb_11721 1338 Cyanobacteria Ascusb_11736 1339 BacteroidesAscusb_11752 1340 Clostridium_XI Ascusb_11753 1341 Clostridium_XIVaAscusb_11757 1342 Ruminococcus Ascusb_11761 1343 SaccharofermentansAscusb_11780 1344 Oscillibacter Ascusb_11781 1345Lachnospiracea_incertae_sedis Ascusb_11783 1346 Fibrobacter Ascusb_117931347 Kiloniella Ascusb_11809 1348 Olivibacter Ascusb_11819 1349Clostridium_IV Ascusb_11821 1350 Spirochaeta Ascusb_11865 1351Prevotella Ascusb_11870 1352 Olivibacter Ascusb_11881 1353 PrevotellaAscusb_11884 1354 Parabacteroides Ascusb_11885 1355 PrevotellaAscusb_11892 1356 Leifsonia Ascusb_11896 1357 Clostridium_IVAscusb_11901 1358 Victivallis Ascusb_11903 1359 Treponema Ascusb_119291360 Cyanobacteria Ascusb_11952 1361 Sporotomaculum Ascusb_11954 1362Spirochaeta Ascusb_11955 1363 Clostridium_III Ascusb_11960 1364Clostridium_XIVa Ascusb_11962 1365 Anaerovorax Ascusb_11963 1366Oscillibacter Ascusb_11964 1367 Victivallis Ascusb_11988 1368Lachnospiracea_incertae_sedis Ascusb_11993 1369 Spirochaeta Ascusb_119971370 Clostridium_XIVb Ascusb_12000 1371 Oscillibacter Ascusb_12004 1372Prevotella Ascusb_12013 1373 Anaeroplasma Ascusb_12046 1374Adlercreutzia Ascusb_12054 1375 Clostridium_XIVa Ascusb_12061 1376Beijerinckia Ascusb_12069 1377 Prevotella Ascusb_12106 1378 CoprococcusAscusb_12110 1379 Lentisphaera Ascusb_12116 1380 Clostridium_XIVaAscusb_12119 1381 Saccharofermentans Ascusb_12127 1382 PorphyrobacterAscusb_12128 1383 Rhodobacter Ascusb_12140 1384 OscillibacterAscusb_12153 1385 Roseburia Ascusb_12160 1386 Prevotella Ascusb_121751387 Aquiflexum Ascusb_12177 1388 Rhodopirellula Ascusb_12187 1389Bacteroides Ascusb_12191 1390 Bacteroides Ascusb_12216 1391Clostridium_XIVa Ascusb_12221 1392 Clostridium_IV Ascusb_12227 1393Prevotella Ascusb_12243 1394 Mogibacterium Ascusb_12248 1395 PrevotellaAscusb_12252 1396 Clostridium_XIVa Ascusb_12269 1397 PrevotellaAscusb_12270 1398 Capnocytophaga Ascusb_12276 1399 AcholeplasmaAscusb_12282 1400 Clostridium_IV Ascusb_12310 1401 SuccinivibrioAscusb_12327 1402 Pseudonocardia Ascusb_12339 1403 Clostridium_XIVaAscusb_12353 1404 Butyricimonas Ascusb_12354 1405 AnaerovoraxAscusb_12355 1406 Prevotella Ascusb_12383 1407 ButyricimonasAscusb_12399 1408 Parabacteroides Ascusb_12407 1409 Clostridium_XIVaAscusb_12413 1410 Clostridium_XIVb Ascusb_12417 1411 BacteroidesAscusb_12428 1412 Cyanobacteria Ascusb_12452 1413 RiemerellaAscusb_12461 1414 Anaeroplasma Ascusb_12487 1415 RuminococcusAscusb_12489 1416 Verrucomicrobia Ascusb_12499 1417Lachnospiracea_incertae_sedis Ascusb_12511 1418 SyntrophococcusAscusb_12512 1419 Clostridium_IV Ascusb_12520 1420 BarnesiellaAscusb_12534 1421 Olivibacter Ascusb_12553 1422 Clostridium_XIVaAscusb_12574 1423 Cryptanaerobacter Ascusb_12577 1424 SaccharofermentansAscusb_12578 1425 Clostridium_IV Ascusb_12599 1426 CoprococcusAscusb_12600 1427 Barnesiella Ascusb_12606 1428Clostridium_sensu_stricto Ascusb_12618 1429 HydrogenoanaerobacteriumAscusb_12627 1430 Clostridium_XIVb Ascusb_12628 1431 SelenomonasAscusb_12661 1432 Prevotella Ascusb_12662 1433 HydrogenoanaerobacteriumAscusb_12679 1434 Spirochaeta Ascusb_12703 1435 EnterorhabdusAscusb_12704 1436 Thermoanaerobacter Ascusb_12709 1437 ArmatimonadetesAscusb_12719 1438 Syntrophococcus Ascusb_12723 1439 SphingobiumAscusb_12731 1440 Clostridium_XIVa Ascusb_12737 1441 GeosporobacterAscusb_12740 1442 Enterorhabdus Ascusb_12746 1443 VerrucomicrobiaAscusb_12747 1444 Clostridium_XIVa Ascusb_12749 1445 ParabacteroidesAscusb_12750 1446 Cryptanaerobacter Ascusb_12769 1447 AnaeroplasmaAscusb_12775 1448 Spirochaeta Ascusb_12779 1449 Prevotella Ascusb_128041450 Roseburia Ascusb_12819 1451 Pedobacter Ascusb_12826 1452 PedobacterAscusb_12835 1453 Eggerthella Ascusb_12838 1454 Prevotella Ascusb_128531455 Rikenella Ascusb_12873 1456 Anaerophaga Ascusb_12894 1457Spirochaeta Ascusb_12901 1458 Clostridium_IV Ascusb_12910 1459 WeissellaAscusb_12931 1460 Butyricicoccus Ascusb_12946 1461 Hahella Ascusb_129531462 Acholeplasma Ascusb_12960 1463 Clostridium_XIVa Ascusb_12962 1464Cellulosilyticum Ascusb_12987 1465 Verrucomicrobia Ascusb_12995 1466Clostridium_XIVa Ascusb_13002 1467 Pseudoflavonifractor Ascusb_130281468 Calditerricola Ascusb_13035 1469 Clostridium_IV Ascusb_13039 1470Clostridium_IV Ascusb_13050 1471 Adlercreutzia Ascusb_13054 1472Bulleidia Ascusb_13088 1473 Lachnospiracea_incertae_sedis Ascusb_130891474 Mucilaginibacter Ascusb_13115 1475 Victivallis Ascusb_13128 1476Anaerovorax Ascusb_13130 1477 Clostridium_XIVb Ascusb_13134 1478Clostridium_XIVa Ascusb_13154 1479 Prevotella Ascusb_13155 1480Bacteroides Ascusb_13163 1481 Schwartzia Ascusb_13165 1482Pyramidobacter Ascusb_13226 1483 Eubacterium Ascusb_13230 1484Lachnospiracea_incertae_sedis Ascusb_13244 1485 Clostridium_XIVaAscusb_13249 1486 Roseburia Ascusb_13254 1487 Clostridium_XIVbAscusb_13276 1488 Enterorhabdus Ascusb_13284 1489 PedobacterAscusb_13291 1490 Clostridium_sensu_stricto Ascusb_13296 1491Clostridium_XIVa Ascusb_13328 1492 Clostridium_III Ascusb_13343 1493Desulfotomaculum Ascusb_13349 1494 Clostridium_IV Ascusb_13353 1495Proteiniclasticum Ascusb_13371 1496 Prevotella Ascusb_13412 1497Faecalibacterium Ascusb_13417 1498 Microbacterium Ascusb_13419 1499Leucobacter Ascusb_13424 1500 Prevotella Ascusb_13426 1501Sphingobacterium Ascusb_13457 1502 Fusibacter Ascusb_13458 1503Howardella Ascusb_13463 1504 Pedobacter Ascusb_13488 1505 CaldilineaAscusb_13504 1506 Turicibacter Ascusb_13513 1507 Clostridium_IVAscusb_13516 1508 Alistipes Ascusb_13546 1509 Clostridium_XIVaAscusb_13547 1510 Clostridium_XIVa Ascusb_13567 1511 PrevotellaAscusb_13597 1512 Clostridium_XIVa Ascusb_13611 1513 ButyricimonasAscusb_13648 1514 Anaerovibrio Ascusb_13663 1515 Prevotella Ascusb_136751516 Pseudoflavonifractor Ascusb_13679 1517 Corynebacterium Ascusb_137631518 Leucobacter Ascusb_13780 1519 Kerstersia Ascusb_13819 1520 SlackiaAscusb_13835 1521 Lactococcus Ascusb_13839 1522 Prevotella Ascusb_138401523 Clostridium_IV Ascusb_13845 1524 Prevotella Ascusb_13848 1525Bacteroides Ascusb_13867 1526 Lactobacillus Ascusb_13881 1527 PrevotellaAscusb_13892 1528 Clostridium_XIVa Ascusb_13895 1529Clostridium_sensu_stricto Ascusb_13903 1530 Syntrophococcus Ascusb_139041531 Clostridium_XIVa Ascusb_13921 1532 Victivallis Ascusb_13923 1533Bacteroides Ascusb_13940 1534 Acidobacteria Ascusb_13951 1535Clostridium_XIVa Ascusb_13953 1536 Prevotella Ascusb_13954 1537Verrucomicrobia Ascusb_13955 1538 Clostridium_XIVa Ascusb_13981 1539Treponema Ascusb_13982 1540 Pyramidobacter Ascusb_13983 1541Robinsoniella Ascusb_13992 1542 Lachnospiracea_incertae_sedisAscusb_13995 1543 Clostridium_XI Ascusb_13996 1544 BifidobacteriumAscusb_14005 1545 Bacteroides Ascusb_14013 1546 GordonibacterAscusb_14016 1547 Enterorhabdus Ascusb_14055 1548 LactobacillusAscusb_14059 1549 Bacteroides Ascusb_14074 1550 Prevotella Ascusb_140861551 Tannerella Ascusb_14141 1552 Bacteroides Ascusb_14145 1553Prevotella Ascusb_14151 1554 Clostridium_XIVb Ascusb_14163 1555Gelidibacter Ascusb_14189 1556 Cyanobacteria Ascusb_14213 1557Rhodoplanes Ascusb_14224 1558 Selenomonas Ascusb_14226 1559Escherichia/Shigella Ascusb_14256 1560 Rikenella Ascusb_14278 1561Coprococcus Ascusb_14285 1562 Clostridium_sensu_stricto Ascusb_142901563 Hyphomicrobium Ascusb_14304 1564 Erysipelotrichaceae_incertae_sedisAscusb_14320 1565 Verrucomicrobia Ascusb_14324 1566 StaphylococcusAscusb_14335 1567 Verrucomicrobia Ascusb_14358 1568 VictivallisAscusb_14359 1569 Selenomonas Ascusb_14423 1570 DesulfobulbusAscusb_14425 1571 Clostridium_III Ascusb_14450 1572 SpirochaetaAscusb_14451 1573 Kordia Ascusb_14514 1574 Bosea Ascusb_14521 1575Enterococcus Ascusb_14525 1576 Clostridium_III Ascusb_14530 1577Xanthobacter Ascusb_14538 1578 Lactobacillus Ascusb_14555 1579Prevotella Ascusb_14583 1580 Acidaminococcus Ascusb_14595 1581Eubacterium Ascusb_14596 1582 Bacteroides Ascusb_14611 1583Clostridium_XIVa Ascusb_14613 1584 Lactobacillus Ascusb_14626 1585Devosia Ascusb_14628 1586 Pedobacter Ascusb_14667 1587 Clostridium_IVAscusb_14747 1588 Clostridium_XIVa Ascusb_14785 1589 CorynebacteriumAscusb_14790 1590 Spirochaeta Ascusb_14792 1591 AnaeroplasmaAscusb_14828 1592 Clostridium_XIVa Ascusb_14869 1593Lachnospiracea_incertae_sedis Ascusb_14888 1594 SaccharofermentansAscusb_14898 1595 Slackia Ascusb_14906 1596 Limibacter Ascusb_14951 1597Sphingobium Ascusb_14952 1598 Clostridium_XIVa Ascusb_14987 1599Riemerella Ascusb_14990 1600 Saccharofermentans Ascusb_15032 1601Bacteroides Ascusb_15048 1602 Prevotella Ascusb_15076 1603 SelenomonasAscusb_15097 1604 Victivallis Ascusb_15122 1605 Howardella Ascusb_151281606 Pelospora Ascusb_15132 1607 Clostridium_sensu_stricto Ascusb_151511608 Selenomonas Ascusb_15156 1609 Fibrobacter Ascusb_15181 1610Clostridium_III Ascusb_15215 1611 Sphingomonas Ascusb_15220 1612Selenomonas Ascusb_15226 1613 Eggerthella Ascusb_15326 1614 TreponemaAscusb_15352 1615 Mogibacterium Ascusb_15357 1616 AdlercreutziaAscusb_15390 1617 Selenomonas Ascusb_15394 1618 MethylomicrobiumAscusb_15404 1619 Leuconostoc Ascusb_15413 1620 PyramidobacterAscusb_15427 1621 Butyrivibrio Ascusb_15438 1622 BacteroidesAscusb_15454 1623 Butyricimonas Ascusb_15455 1624 RuminococcusAscusb_15461 1625 Clostridium_sensu_stricto Ascusb_15482 1626Butyrivibrio Ascusb_15488 1627 Corynebacterium Ascusb_15494 1628Proteiniborus Ascusb_15526 1629 Spirochaeta Ascusb_15539 1630Acetitomaculum Ascusb_15549 1631 Selenomonas Ascusb_15552 1632Altererythrobacter Ascusb_15556 1633 Atopobium Ascusb_15587 1634Clostridium_IV Ascusb_15615 1635 Clostridium_XIVa Ascusb_15624 1636Clostridium_XIVa Ascusb_15695 1637 Clostridium_IV Ascusb_15703 1638Clostridium_III Ascusb_15720 1639 Candidate phylumTM7 Ascusb_15737 1640Desulfotomaculum Ascusb_15741 1641 Pedobacter Ascusb_15746 1642Bacteroides Ascusb_15750 1643 Asaccharobacter Ascusb_15754 1644Microbacterium Ascusb_15768 1645 Treponema Ascusb_15824 1646Dethiosulfovibrio Ascusb_15830 1647 Oscillibacter Ascusb_15832 1648Selenomonas Ascusb_15846 1649 Eubacterium Ascusb_15864 1650 RuminococcusAscusb_15877 1651 Treponema Ascusb_15915 1652 Spirochaeta Ascusb_159511653 Roseburia Ascusb_15963 1654 Ruminococcus Ascusb_15992 1655Butyricimonas Ascusb_16010 1656 Pedobacter Ascusb_16051 1657 SpirochaetaAscusb_16066 1658 Parabacteroides Ascusb_16101 1659 MethylococcusAscusb_16111 1660 Enterorhabdus Ascusb_16113 1661Clostridium_sensu_stricto Ascusb_16124 1662 Gelidibacter Ascusb_161491663 Sporobacter Ascusb_16168 1664 Pedobacter Ascusb_16185 1665Cyanobacteria Ascusb_16194 1666 Syntrophococcus Ascusb_16198 1667Slackia Ascusb_16200 1668 Mogibacterium Ascusb_16215 1669 PrevotellaAscusb_16239 1670 Pseudoflavonifractor Ascusb_16244 1671 VeillonellaAscusb_16257 1672 Clostridium_XIVa Ascusb_16278 1673 BacillusAscusb_16299 1674 Pedobacter Ascusb_16316 1675 Clostridium_IVAscusb_16329 1676 Fibrobacter Ascusb_16330 1677 PaenibacillusAscusb_16336 1678 Brevundimonas Ascusb_16345 1679 DesulfovibrioAscusb_16373 1680 Clostridium_XI Ascusb_16374 1681 HelicobacterAscusb_16383 1682 Prevotella Ascusb_16420 1683 Clostridium_XIVaAscusb_16423 1684 Prevotella Ascusb_16436 1685 Herbiconiux Ascusb_164531686 Clostridium_IV Ascusb_16461 1687 Rikenella Ascusb_16470 1688Clostridium_XIVa Ascusb_16473 1689 Hippea Ascusb_16536 1690Lactobacillus Ascusb_16537 1691 Eubacterium Ascusb_16541 1692Clostridium_IV Ascusb_16546 1693 Clostridium_III Ascusb_16560 1694Lactobacillus Ascusb_16565 1695 Lactobacillus Ascusb_16574 1696Desulfotomaculum Ascusb_16578 1697 Prevotella Ascusb_16618 1698Staphylococcus Ascusb_16628 1699 Tenacibaculum Ascusb_16632 1700Parabacteroides Ascusb_16655 1701 Clostridium_XIVa Ascusb_16668 1702Clostridium_IV Ascusb_16671 1703 Clostridium_IV Ascusb_16674 1704Pedobacter Ascusb_16682 1705 Helicobacter Ascusb_16686 1706Proteiniclasticum Ascusb_16691 1707 Anaplasma Ascusb_16711 1708Bacteroides Ascusb_16734 1709 Clostridium_IV Ascusb_16749 1710Mucilaginibacter Ascusb_16803 1711 Verrucomicrobia Ascusb_16829 1712Selenomonas Ascusb_16884 1713 Parabacteroides Ascusb_16931 1714Eubacterium Ascusb_16933 1715 Coprococcus Ascusb_16948 1716 WeissellaAscusb_16968 1717 Pedobacter Ascusb_16992 1718 Clostridium_XIAscusb_16995 1719 Sphingomonas Ascusb_16998 1720 Treponema Ascusb_170131721 Geobacter Ascusb_17017 1722 Clostridium_XIVa Ascusb_17018 1723Filomicrobium Ascusb_17036 1724 Prevotella Ascusb_17038 1725 PedobacterAscusb_17057 1726 Pedobacter Ascusb_17058 1727 Clostridium_XIVaAscusb_17064 1728 Bifidobacterium Ascusb_17066 1729 SaccharofermentansAscusb_17092 1730 Ruminococcus Ascusb_17136 1731 FlavobacteriumAscusb_17138 1732 Rhodopirellula Ascusb_17161 1733 RoseburiaAscusb_17171 1734 Prevotella Ascusb_17177 1735 Limibacter Ascusb_171821736 Saccharofermentans Ascusb_17203 1737 Clostridium_sensu_strictoAscusb_17206 1738 Clostridium_III Ascusb_17243 1739 PrevotellaAscusb_17275 1740 Pseudoxanthomonas Ascusb_17283 1741 AnaerorhabdusAscusb_17325 1742 Clostridium_III Ascusb_17360 1743 StreptomycesAscusb_17372 1744 Pedobacter Ascusb_17388 1745 Cellulomonas Ascusb_174141746 Clostridium_XIVa Ascusb_17416 1747 Olivibacter Ascusb_17425 1748Treponema Ascusb_17433 1749 Gelidibacter Ascusb_17437 1750 RuminococcusAscusb_17439 1751 Clostridium_IV Ascusb_17446 1752 GemmatimonasAscusb_17450 1753 Prevotella Ascusb_17459 1754 EthanoligenensAscusb_17477 1755 Leucobacter Ascusb_17494 1756 Clostridium_XIVaAscusb_17502 1757 Clostridium_XIVa Ascusb_17507 1758 EggerthellaAscusb_17540 1759 Prevotella Ascusb_17553 1760 Prevotella Ascusb_175691761 Solobacterium Ascusb_17571 1762 Xanthobacter Ascusb_17581 1763Verrucomicrobia Ascusb_17649 1764 Desulfovibrio Ascusb_17670 1765Microbacterium Ascusb_17717 1766 Oscillibacter Ascusb_17718 1767 BlautiaAscusb_17735 1768 Papillibacter Ascusb_17736 1769 PrevotellaAscusb_17759 1770 Lentisphaera Ascusb_17766 1771 RuminococcusAscusb_17767 1772 Bacteroides Ascusb_17769 1773 Catonella Ascusb_177711774 Clostridium_XIVa Ascusb_17773 1775 Clostridium_IV Ascusb_17782 1776Verrucomicrobia Ascusb_17802 1777 Clostridium_XI Ascusb_17804 1778Prevotella Ascusb_17810 1779 Candidate phylumTM7 Ascusb_17824 1780Mogibacterium Ascusb_17838 1781 Clostridium_XIVa Ascusb_17846 1782Ruminococcus Ascusb_17857 1783 Eubacterium Ascusb_17866 1784Clostridium_IV Ascusb_17892 1785 Rhodomicrobium Ascusb_17896 1786Butyricicoccus Ascusb_17957 1787 Saccharofermentans Ascusb_17975 1788Prevotella Ascusb_17978 1789 Mannheimia Ascusb_17981 1790 LactobacillusAscusb_18078 1791 Clostridium_IV Ascusb_18081 1792 Clostridium_IVAscusb_18091 1793 Adlercreutzia Ascusb_18107 1794 SelenomonasAscusb_18110 1795 Paenibacillus Ascusb_18123 1796 Clostridium_IVAscusb_18140 1797 Paenibacillus Ascusb_18148 1798 ButyricimonasAscusb_18161 1799 Wandonia Ascusb_18170 1800 Puniceicoccus Ascusb_181791801 Lactonifactor Ascusb_18183 1802 Selenomonas Ascusb_18248 1803Brevundimonas Ascusb_18262 1804 Prevotella Ascusb_18273 1805Gelidibacter Ascusb_18283 1806 Mogibacterium Ascusb_18287 1807Clostridium_XIVa Ascusb_18303 1808 Coprococcus Ascusb_18329 1809Verrucomicrobia Ascusb_18335 1810 Barnesiella Ascusb_18339 1811Verrucomicrobia Ascusb_18351 1812 Clostridium_XIVa Ascusb_18354 1813Anaerovorax Ascusb_18371 1814 Bacteroides Ascusb_18389 1815Parasporobacterium Ascusb_18444 1816 Prevotella Ascusb_18449 1817Parapedobacter Ascusb_18475 1818 Streptomyces Ascusb_18495 1819Candidate phylumTM7 Ascusb_18503 1820 Thermotalea Ascusb_18516 1821Alkaliflexus Ascusb_18519 1822 Oscillibacter Ascusb_18557 1823Anaerotruncus Ascusb_18564 1824 Spirochaeta Ascusb_18566 1825Clostridium_XI Ascusb_18567 1826 Sporotomaculum Ascusb_18585 1827Sporacetigenium Ascusb_18592 1828 Bulleidia Ascusb_18608 1829Clostridium_IV Ascusb_18636 1830 Syntrophomonas Ascusb_18648 1831Desulfatiferula Ascusb_18678 1832 Hydrogenoanaerobacterium Ascusb_186801833 Clostridium_XIVa Ascusb_18695 1834 Mogibacterium Ascusb_18731 1835Spirochaeta Ascusb_18733 1836 Prevotella Ascusb_18735 1837 TreponemaAscusb_18738 1838 Spiroplasma Ascusb_18764 1839 Clostridium_XIVaAscusb_18766 1840 Bacteroides Ascusb_18795 1841 Treponema Ascusb_188141842 Selenomonas Ascusb_18829 1843 Butyricicoccus Ascusb_18846 1844Gelidibacter Ascusb_18866 1845 Acetitomaculum Ascusb_18876 1846Proteiniclasticum Ascusb_18907 1847 Papillibacter Ascusb_18930 1848Prevotella Ascusb_18949 1849 Elusimicrobium Ascusb_18970 1850Lachnospiracea_incertae_sedis Ascusb_18998 1851 Devosia Ascusb_190061852 Roseburia Ascusb_19052 1853 Mucilaginibacter Ascusb_19054 1854Mogibacterium Ascusb_19056 1855 Saccharofermentans Ascusb_19063 1856Paenibacillus Ascusb_19092 1857 Anaerotruncus Ascusb_19101 1858Leucobacter Ascusb_19114 1859 Clostridium_XIVa Ascusb_19148 1860Eubacterium Ascusb_19160 1861 Beijerinckia Ascusb_19170 1862 PrevotellaAscusb_19200 1863 Clostridium_III Ascusb_19206 1864 CyanobacteriaAscusb_19219 1865 Pseudoflavonifractor Ascusb_19237 1866 ButyrivibrioAscusb_19245 1867 Acholeplasma Ascusb_19267 1868 FilomicrobiumAscusb_19288 1869 Clostridium_III Ascusb_19335 1870 PseudoflavonifractorAscusb_19340 1871 Anaerophaga Ascusb_19341 1872Lachnospiracea_incertae_sedis Ascusb_19347 1873 AsaccharobacterAscusb_19353 1874 Kordia Ascusb_19371 1875 Ruminococcus Ascusb_193761876 Clostridium_III Ascusb_19379 1877 Ethanoligenens Ascusb_19392 1878Clostridium_XIVa Ascusb_19412 1879 Barnesiella Ascusb_19414 1880Eubacterium Ascusb_19444 1881 Prevotella Ascusb_19457 1882 AnaerophagaAscusb_19496 1883 Acetitomaculum Ascusb_19498 1884 PrevotellaAscusb_19503 1885 Clostridium_III Ascusb_19507 1886 MarinoscillumAscusb_19558 1887 Pedobacter Ascusb_19568 1888 Prevotella Ascusb_195791889 Prevotella Ascusb_19613 1890 Anaerovorax Ascusb_19633 1891Clostridium_XIVa Ascusb_19658 1892 Clostridium_IV Ascusb_19662 1893Lachnospiracea_incertae_sedis Ascusb_19681 1894Clostridium_sensu_stricto Ascusb_19694 1895 Lishizhenia Ascusb_196981896 Pedobacter Ascusb_19700 1897 Howardella Ascusb_19731 1898 RoseburiaAscusb_19745 1899 Clostridium_XIVa Ascusb_19754 1900 AnaerovoraxAscusb_19765 1901 Lentisphaera Ascusb_19772 1902 Prevotella Ascusb_197781903 Saccharofermentans Ascusb_19779 1904 Cyanobacteria Ascusb_198181905 Proteiniphilum Ascusb_19824 1906 Schwartzia Ascusb_19855 1907Anaerorhabdus Ascusb_19884 1908 Robinsoniella Ascusb_19885 1909Clostridium_IV Ascusb_19904 1910 Erysipelotrichaceae_incertae_sedisAscusb_19936 1911 Flavobacterium Ascusb_19950 1912 PedobacterAscusb_19955 1913 Clostridium_III Ascusb_19982 1914 SelenomonasAscusb_20001 1915 Rhizobium Ascusb_20027 1916 Victivallis Ascusb_200441917 Butyricimonas Ascusb_20062 1918 Parabacteroides Ascusb_20064 1919Adhaeribacter Ascusb_20067 1920 Eubacterium Ascusb_20086 1921Acidobacteria Ascusb_20100 1922 Treponema Ascusb_20104 1923Clostridium_XIVa Ascusb_20108 1924 Clostridium_XIVa Ascusb_20135 1925Schwartzia Ascusb_20143 1926 Prevotella Ascusb_20162 1927 SelenomonasAscusb_20172 1928 Beijerinckia Ascusb_20219 1929 EubacteriumAscusb_20221 1930 Adhaeribacter Ascusb_20251 1931 VerrucomicrobiaAscusb_20264 1932 Desulfobulbus Ascusb_20275 1933 BacteroidesAscusb_20278 1934 Rummeliibacillus Ascusb_20291 1935 AgarivoransAscusb_20293 1936 Clostridium_XIVa Ascusb_20306 1937 SelenomonasAscusb_20312 1938 Verrucomicrobia Ascusb_20365 1939 PrevotellaAscusb_20368 1940 Spirochaeta Ascusb_20392 1941 Selenomonas Ascusb_204051942 Spiroplasma Ascusb_20424 1943 Pedobacter Ascusb_20440 1944Clostridium_XIVa Ascusb_20449 1945 Cyanobacteria Ascusb_20456 1946Lactobacillus Ascusb_20463 1947 Clostridium_XIVa Ascusb_20529 1948Prevotella Ascusb_20534 1949 Prevotella Ascusb_20540 1950 MarinobacterAscusb_20569 1951 Butyricimonas Ascusb_20576 1952 PrevotellaAscusb_20594 1953 Dongia Ascusb_20595 1954 Anaerovorax Ascusb_20639 1955Butyricimonas Ascusb_20757 1956 Cryptanaerobacter Ascusb_20826 1957Papillibacter Ascusb_20904 1958 Clostridium_sensu_stricto Ascusb_209381959 Escherichia/Shigella Ascusb_20943 1960 Butyricicoccus Ascusb_209861961 Prevotella Ascusb_21013 1962 Lachnospiracea_incertae_sedisAscusb_21027 1963 Thermotalea Ascusb_21035 1964 CohaesibacterAscusb_21042 1965 Clostridium_XVIII Ascusb_21043 1966Lachnospiracea_incertae_sedis Ascusb_21085 1967 Spirochaeta Ascusb_210951968 Clostridium_XIVa Ascusb_21112 1969 HydrogenoanaerobacteriumAscusb_21147 1970 Clostridium_IV Ascusb_21151 1971 PapillibacterAscusb_21160 1972 Sporosarcina Ascusb_21190 1973 SelenomonasAscusb_21219 1974 Papillibacter Ascusb_21229 1975Lachnospiracea_incertae_sedis Ascusb_21244 1976 Clostridium_XIVaAscusb_21271 1977 Saccharofermentans Ascusb_21297 1978 Clostridium_IVAscusb_21309 1979 Lachnospiracea_incertae_sedis Ascusb_21348 1980Clostridium_IV Ascusb_21425 1981 Lachnospiracea_incertae_sedisAscusb_21436 1982 Desulfotomaculum Ascusb_21466 1983 PedobacterAscusb_21484 1984 Anaeroplasma Ascusb_21546 1985 Clostridium_IVAscusb_21585 1986 Treponema Ascusb_21595 1987 Mogibacterium Ascusb_216011988

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a general workflow of one embodiment of the method fordetermining the absolute abundance of one or more active microorganismstrains.

FIG. 2 shows a general workflow of one embodiment of a method fordetermining the co-occurrence of one or more, or two or more, activemicroorganism strains in a sample with one or more metadata(environmental) parameters, followed by leveraging cluster analysis andcommunity detection methods on the network of determined relationships.

FIG. 3A, FIG. 3B, and FIG. 3C depict the results of a field trial inwhich dairy cows were administered a composition comprising Ascusb_3138and Ascusf_15; FIG. 3A reveals the average number of pounds of milk fatproduced over time; FIG. 3B reveals the average number of pounds of milkprotein produced over time; and FIG. 3C reveals the average number ofpounds of energy corrected milk (ECM) produced over time. The verticalline intersecting the data points in each of FIG. 3A, FIG. 3B, and FIG.3C marks the day at which administration of the microbial bioconsortiaceased.

FIG. 4 depicts the milk yield (kg) daily means (no fill) and covariateadjusted weekly least square means (solid fill)±SEM of cows assignedeither to Control (circle) or Inoculated (trapezoid) by interventionperiod study days.

FIG. 5 depicts the milk crude protein yield (CP, kg) daily means (nofill) and weekly least square means (solid fill)±SEM of cows assignedeither to Control (circle) or Inoculated (trapezoid) by Interventionperiod study days.

FIG. 6 depicts the milk fat yield (kg) daily means (no fill) and weeklyleast square means (solid fill)±SEM of cows assigned either to Control(circle) or Inoculated (trapezoid) by Intervention period study days.

FIG. 7 depicts the energy corrected milk yield (ECM, kg) daily means (nofill) and weekly least square means (solid fill)±SEM of cows assignedeither to Control (circle) or Inoculated (trapezoid) by Interventionperiod study days.

FIG. 8A and FIG. 8B depict the shared percent similarity (percentidentity) among the bacteria (FIG. 8A) and fungi (FIG. 8B) of Table 1.The data points represent the greatest percent similarity pairing foreach strain.

FIG. 9 depicts the MIC score distribution for rumen bacteria and milkfat efficiency.

FIG. 10 depicts the MIC score distribution for rumen fungi and milk fatefficiency.

FIG. 11 depicts the MIC score distribution for rumen bacteria and dairyefficiency.

FIG. 12 depicts the MIC score distribution for rumen fungi and dairyefficiency.

FIG. 13 depicts the MIC score distribution for rumen bacteria and milkfat efficiency with four species of bacteria and their MIC scores, inwhich the species have been evaluated in 3^(rd) party studies. The lowerthe MIC score, the less likely the species/strains are capable ofpositively modulating milk fat efficiency in dairy cows.

FIG. 14 depicts an undegraded carbon source (Day 0) and a degradedcarbon source (Day 7), as utilized in the insoluble carbon sourceassays.

FIG. 15 depicts a decrease in the number of cows exhibiting greater than200,000 somatic cell counts (SSC)/mL milk in dairy cows that wereadministered a microbial compostion of the present disclosure versusdairy cows that were not administered a microbial composition of thepresent disclosure.

FIG. 16 depicts a diagram that exemplifies how the diet influences theproduction of volatile fatty acids which in turn modulate milkproduction, body condition, growth, etc. Reproduced from Moran, 2005.Tropical dairy farming: feeding management for small holder dairyfarmers in the humic tropics (Chapter 5), Landlinks Press, 312 pp.

FIG. 17 depicts a schematic diagram that illustrates an example processflow for use with an exemplary microbe interaction analysis andselection system, including the determination of MIC scores discussedthroughout the present disclosure.

FIG. 18 depicts the non-linearity of pounds of milk fate produced overthe course of an experiment to determine rumen microbial communityconstituents that impact the production of milk fat in dairy cows.

FIG. 19 depicts the correlation of the absolute cell count with activityfilter of target strain Ascus_713 to pounds (lbs) of milk fat produced.

FIG. 20 depicts the absolute cell count with activity filter of targetstrain Ascus_7 and the pounds (lbs) of milk fat produced over the courseof an experiment.

FIG. 21 depicts the correlation of the relative abundance with noactivity filter of target strain Ascus_3038 to pounds (lbs) of milk fatproduced.

DETAILED DESCRIPTION Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

The term “a” or “an” may refer to one or more of that entity, i.e. canrefer to plural referents. As such, the terms “a” or “an”, “one or more”and “at least one” are used interchangeably herein. In addition,reference to “an element” by the indefinite article “a” or “an” does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there is one and only one ofthe elements.

Reference throughout this specification to “one embodiment”, “anembodiment”, “one aspect”, or “an aspect” means that a particularfeature, structure or characteristic described in connection with theembodiment is included in at least one embodiment of the presentdisclosure. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics can be combined inany suitable manner in one or more embodiments.

As used herein, in particular embodiments, the terms “about” or“approximately” when preceding a numerical value indicates the valueplus or minus a range of 10%.

As used herein the terms “microorganism” or “microbe” should be takenbroadly. These terms are used interchangeably and include, but are notlimited to, the two prokaryotic domains, Bacteria and Archaea,eukaryotic fungi and protists, as well as viruses. In some embodiments,the disclosure refers to the “microbes” of Table 1 or Table 3, or the“microbes” incorporated by reference. This characterization can refer tonot only the predicted taxonomic microbial identifiers of the table, butalso the identified strains of the microbes listed in the table.

The term “microbial consortia” or “microbial consortium” refers to asubset of a microbial community of individual microbial species, orstrains of a species, which can be described as carrying out a commonfunction, or can be described as participating in, or leading to, orcorrelating with, a recognizable parameter, such as a phenotypic traitof interest (e.g. increased milk production in a ruminant). Thecommunity may comprise two or more species, or strains of a species, ofmicrobes. In some instances, the microbes coexist within the communitysymbiotically.

The term “microbial community” means a group of microbes comprising twoor more species or strains. Unlike microbial consortia, a microbialcommunity does not have to be carrying out a common function, or doesnot have to be participating in, or leading to, or correlating with, arecognizable parameter, such as a phenotypic trait of interest (e.g.increased milk production in a ruminant).

As used herein, “isolate,” “isolated,” “isolated microbe,” and liketerms, are intended to mean that the one or more microorganisms has beenseparated from at least one of the materials with which it is associatedin a particular environment (for example soil, water, animal tissue).

Microbes of the present disclosure may include spores and/or vegetativecells. In some embodiments, microbes of the present disclosure includemicrobes in a viable but non-culturable (VBNC) state. See Liao and Zhao(US Publication US2015267163A1). In some embodiments, microbes of thepresent disclosure include microbes in a biofilm. See Merritt et al.(U.S. Pat. No. 7,427,408).

Thus, an “isolated microbe” does not exist in its naturally occurringenvironment; rather, it is through the various techniques describedherein that the microbe has been removed from its natural setting andplaced into a non-naturally occurring state of existence. Thus, theisolated strain or isolated microbe may exist as, for example, abiologically pure culture, or as spores (or other forms of the strain)in association with an acceptable carrier.

As used herein, “spore” or “spores” refer to structures produced bybacteria and fungi that are adapted for survival and dispersal. Sporesare generally characterized as dormant structures, however spores arecapable of differentiation through the process of germination.Germination is the differentiation of spores into vegetative cells thatare capable of metabolic activity, growth, and reproduction. Thegermination of a single spore results in a single fungal or bacterialvegetative cell. Fungal spores are units of asexual reproduction, and insome cases are necessary structures in fungal life cycles. Bacterialspores are structures for surviving conditions that may ordinarily benonconductive to the survival or growth of vegetative cells.

As used herein, “microbial composition” refers to a compositioncomprising one or more microbes of the present disclosure, wherein amicrobial composition, in some embodiments, is administered to animalsof the present disclosure.

As used herein, “carrier”, “acceptable carrier”, or “pharmaceuticalcarrier” refers to a diluent, adjuvant, excipient, or vehicle with whichthe compound is administered. Such carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable, orsynthetic origin; such as peanut oil, soybean oil, mineral oil, sesameoil, and the like. Water or aqueous solution saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, in some embodiments as injectable solutions. Alternatively,the carrier can be a solid dosage form carrier, including but notlimited to one or more of a binder (for compressed pills), a glidant, anencapsulating agent, a flavorant, and a colorant. The choice of carriercan be selected with regard to the intended route of administration andstandard pharmaceutical practice. See Hardee and Baggo (1998.Development and Formulation of Veterinary Dosage Forms. 2^(nd) Ed. CRCPress. 504 pg.); E. W. Martin (1970. Remington's PharmaceuticalSciences. 17^(th) Ed. Mack Pub. Co.); and Blaser et al. (US PublicationUS20110280840A1).

In certain aspects of the disclosure, the isolated microbes exist asisolated and biologically pure cultures. It will be appreciated by oneof skill in the art, that an isolated and biologically pure culture of aparticular microbe, denotes that said culture is substantially free(within scientific reason) of other living organisms and contains onlythe individual microbe in question. The culture can contain varyingconcentrations of said microbe. The present disclosure notes thatisolated and biologically pure microbes often “necessarily differ fromless pure or impure materials.” See, e.g. In re Bergstrom, 427 F.2d1394, (CCPA 1970)(discussing purified prostaglandins), see also, In reBergy, 596 F.2d 952 (CCPA 1979)(discussing purified microbes), see also,Parke-Davis & Co. v. H.K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911)(Learned Hand discussing purified adrenaline), aff'd in part, rev'd inpart, 196 F. 496 (2d Cir. 1912), each of which are incorporated hereinby reference. Furthermore, in some aspects, the disclosure provides forcertain quantitative measures of the concentration, or puritylimitations, that must be found within an isolated and biologically puremicrobial culture. The presence of these purity values, in certainembodiments, is a further attribute that distinguishes the presentlydisclosed microbes from those microbes existing in a natural state. See,e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4thCir. 1958) (discussing purity limitations for vitamin B12 produced bymicrobes), incorporated herein by reference.

As used herein, “individual isolates” should be taken to mean acomposition, or culture, comprising a predominance of a single genera,species, or strain, of microorganism, following separation from one ormore other microorganisms. The phrase should not be taken to indicatethe extent to which the microorganism has been isolated or purified.However, “individual isolates” can comprise substantially only onegenus, species, or strain, of microorganism.

As used herein, “microbiome” refers to the collection of microorganismsthat inhabit the digestive tract or gastrointestinal tract of an animal(including the rumen if said animal is a ruminant) and themicroorgansims' physical environment (i.e. the microbiome has a bioticand physical component). The microbiome is fluid and may be modulated bynumerous naturally occurring and artificial conditions (e.g., change indiet, disease, antimicrobial agents, influx of additionalmicroorganisms, etc.). The modulation of the microbiome of a rumen thatcan be achieved via administration of the compositions of thedisclosure, can take the form of: (a) increasing or decreasing aparticular Family, Genus, Species, or functional grouping of microbe(i.e. alteration of the biotic component of the rumen microbiome) and/or(b) increasing or decreasing volatile fatty acids in the rumen,increasing or decreasing rumen pH, increasing or decreasing any otherphysical parameter important for rumen health (i.e. alteration of theabiotic component of the rumen mircrobiome).

As used herein, “probiotic” refers to a substantially pure microbe(i.e., a single isolate) or a mixture of desired microbes, and may alsoinclude any additional components that can be administered to a mammalfor restoring microbiota. Probiotics or microbial inoculant compositionsof the invention may be administered with an agent to allow the microbesto survive the environment of the gastrointestinal tract, i.e., toresist low pH and to grow in the gastrointestinal environment. In someembodiments, the present compositions (e.g., microbial compositions) areprobiotics in some aspects.

As used herein, “prebiotic” refers to an agent that increases the numberand/or activity of one or more desired microbes. Non-limiting examplesof prebiotics that may be useful in the methods of the presentdisclosure include fructooligosaccharides (e.g., oligofructose, inulin,inulin-type fructans), galactooligosaccharides, amino acids, alcohols,and mixtures thereof. See Ramirez-Farias et al. (2008. Br. J. Nutr.4:1-10) and Pool-Zobel and Sauer (2007. J. Nutr. 137:2580-2584 andsupplemental).

The term “growth medium” as used herein, is any medium which is suitableto support growth of a microbe. By way of example, the media may benatural or artificial including gastrin supplemental agar, LB media,blood serum, and tissue culture gels. It should be appreciated that themedia may be used alone or in combination with one or more other media.It may also be used with or without the addition of exogenous nutrients.

The medium may be amended or enriched with additional compounds orcomponents, for example, a component which may assist in the interactionand/or selection of specific groups of microorganisms. For example,antibiotics (such as penicillin) or sterilants (for example, quaternaryammonium salts and oxidizing agents) could be present and/or thephysical conditions (such as salinity, nutrients (for example organicand inorganic minerals (such as phosphorus, nitrogenous salts, ammonia,potassium and micronutrients such as cobalt and magnesium), pH, and/ortemperature) could be amended.

As used herein, the term “ruminant” includes mammals that are capable ofacquiring nutrients from plant-based food by fermenting it in aspecialized stomach (rumen) prior to digestion, principally throughmicrobial actions. Ruminants included cattle, goats, sheep, giraffes,yaks, deer, antelope, and others.

As used herein, the term “bovid” includes any member of family Bovidae,which include hoofed mammals such as antelope, sheep, goats, and cattle,among others.

As used herein, “energy-corrected milk” or “ECM” represents the amountof energy in milk based upon milk volume, milk fat, and milk protein.ECM adjusts the milk components to 3.5% fat and 3.2% protein, thusequalizing animal performance and allowing for comparison of productionat the individual animal and herd levels over time. An equation used tocalculate ECM, as related to the present disclosure, is:

ECM=(0.327×milk pounds)+(12.95×fat pounds)+(7.2×protein pounds)

As used herein, “improved” should be taken broadly to encompassimprovement of a characteristic of interest, as compared to a controlgroup, or as compared to a known average quantity associated with thecharacteristic in question. For example, “improved” milk productionassociated with application of a beneficial microbe, or consortia, ofthe disclosure can be demonstrated by comparing the milk produced by anungulate treated by the microbes taught herein to the milk of anungulate not treated. In the present disclosure, “improved” does notnecessarily demand that the data be statistically significant (i.e.p<0.05); rather, any quantifiable difference demonstrating that onevalue (e.g. the average treatment value) is different from another (e.g.the average control value) can rise to the level of “improved.”

As used herein, “inhibiting and suppressing” and like terms should notbe construed to require complete inhibition or suppression, althoughthis may be desired in some embodiments.

The term “marker” or “unique marker” as used herein is an indicator ofunique microorganism type, microorganism strain or activity of amicroorganism strain. A marker can be measured in biological samples andincludes without limitation, a nucleic acid-based marker such as aribosomal RNA gene, a peptide- or protein-based marker, and/or ametabolite or other small molecule marker.

The term “metabolite” as used herein is an intermediate or product ofmetabolism. A metabolite in one embodiment is a small molecule.Metabolites have various functions, including in fuel, structural,signaling, stimulatory and inhibitory effects on enzymes, as a cofactorto an enzyme, in defense, and in interactions with other organisms (suchas pigments, odorants and pheromones). A primary metabolite is directlyinvolved in normal growth, development and reproduction. A secondarymetabolite is not directly involved in these processes but usually hasan important ecological function. Examples of metabolites include butare not limited to antibiotics and pigments such as resins and terpenes,etc. Some antibiotics use primary metabolites as precursors, such asactinomycin which is created from the primary metabolite, tryptophan.Metabolites, as used herein, include small, hydrophilic carbohydrates;large, hydrophobic lipids and complex natural compounds.

As used herein, the term “genotype” refers to the genetic makeup of anindividual cell, cell culture, tissue, organism, or group of organisms.

As used herein, the term “allele(s)” means any of one or morealternative forms of a gene, all of which alleles relate to at least onetrait or characteristic. In a diploid cell, the two alleles of a givengene occupy corresponding loci on a pair of homologous chromosomes.Since the present disclosure, in embodiments, relates to QTLs, i.e.genomic regions that may comprise one or more genes or regulatorysequences, it is in some instances more accurate to refer to “haplotype”(i.e. an allele of a chromosomal segment) instead of “allele”, however,in those instances, the term “allele” should be understood to comprisethe term “haplotype”. Alleles are considered identical when they expressa similar phenotype. Differences in sequence are possible but notimportant as long as they do not influence phenotype.

As used herein, the term “locus” (loci plural) means a specific place orplaces or a site on a chromosome where for example a gene or geneticmarker is found.

As used herein, the term “genetically linked” refers to two or moretraits that are co-inherited at a high rate during breeding such thatthey are difficult to separate through crossing.

A “recombination” or “recombination event” as used herein refers to achromosomal crossing over or independent assortment. The term“recombinant” refers to an organism having a new genetic makeup arisingas a result of a recombination event.

As used herein, the term “molecular marker” or “genetic marker” refersto an indicator that is used in methods for visualizing differences incharacteristics of nucleic acid sequences. Examples of such indicatorsare restriction fragment length polymorphism (RFLP) markers, amplifiedfragment length polymorphism (AFLP) markers, single nucleotidepolymorphisms (SNPs), insertion mutations, microsatellite markers(SSRs), sequence-characterized amplified regions (SCARs), cleavedamplified polymorphic sequence (CAPS) markers or isozyme markers orcombinations of the markers described herein which defines a specificgenetic and chromosomal location. Markers further include polynucleotidesequences encoding 16S or 18S rRNA, and internal transcribed spacer(ITS) sequences, which are sequences found between small-subunit andlarge-subunit rRNA genes that have proven to be especially useful inelucidating relationships or distinctions among when compared againstone another. Mapping of molecular markers in the vicinity of an alleleis a procedure which can be performed by the average person skilled inmolecular-biological techniques.

The primary structure of major rRNA subunit 16S comprise a particularcombination of conserved, variable, and hypervariable regions thatevolve at different rates and enable the resolution of both very ancientlineages such as domains, and more modern lineages such as genera. Thesecondary structure of the 16S subunit include approximately 50 heliceswhich result in base pairing of about 67% of the residues. These highlyconserved secondary structural features are of great functionalimportance and can be used to ensure positional homology in multiplesequence alignments and phylogenetic analysis. Over the previous fewdecades, the 16S rRNA gene has become the most sequenced taxonomicmarker and is the cornerstone for the current systematic classificationof bacteria and archaea (Yarza et al. 2014. Nature Rev. Micro.12:635-45).

A sequence identity of 94.5% or lower for two 16S rRNA genes is strongevidence for distinct genera, 86.5% or lower is strong evidence fordistinct families, 82% or lower is strong evidence for distinct orders,78.5% is strong evidence for distinct classes, and 75% or lower isstrong evidence for distinct phyla. The comparative analysis of 16S rRNAgene sequences enables the establishment of taxonomic thresholds thatare useful not only for the classification of cultured microorganismsbut also for the classification of the many environmental sequences.Yarza et al. 2014. Nature Rev. Micro. 12:635-45).

As used herein, the term “trait” refers to a characteristic orphenotype. For example, in the context of some embodiments of thepresent disclosure, quantity of milk fat produced relates to the amountof triglycerides, triacylglycerides, diacylglycerides,monoacylglycerides, phospholipids, cholesterol, glycolipids, and fattyacids present in milk. Desirable traits may also include other milkcharacteristics, including but not limited to: predominance of shortchain fatty acids, medium chain fatty acids, and long chain fatty acids;quantity of carbohydrates such as lactose, glucose, galactose, and otheroligosaccharides; quantity of proteins such as caseins and whey;quantity of vitamins, minerals, milk yield/volume; reductions in methaneemissions or manure; improved efficiency of nitrogen utilization;improved dry matter intake; improved feed efficiency and digestibility;increased degradation of cellulose, lignin, and hemicellulose; increasedrumen concentrations of fatty acids such as acetic acid, propionic acid,and butyric acid; etc.

A trait may be inherited in a dominant or recessive manner, or in apartial or incomplete-dominant manner. A trait may be monogenic (i.e.determined by a single locus) or polygenic (i.e. determined by more thanone locus) or may also result from the interaction of one or more geneswith the environment.

In the context of this disclosure, traits may also result from theinteraction of one or more mammalian genes and one or more microorganismgenes.

As used herein, the term “homozygous” means a genetic condition existingwhen two identical alleles reside at a specific locus, but arepositioned individually on corresponding pairs of homologous chromosomesin the cell of a diploid organism. Conversely, as used herein, the term“heterozygous” means a genetic condition existing when two differentalleles reside at a specific locus, but are positioned individually oncorresponding pairs of homologous chromosomes in the cell of a diploidorganism.

As used herein, the term “phenotype” refers to the observablecharacteristics of an individual cell, cell culture, organism (e.g., aruminant), or group of organisms which results from the interactionbetween that individual's genetic makeup (i.e., genotype) and theenvironment.

As used herein, the term “chimeric” or “recombinant” when describing anucleic acid sequence or a protein sequence refers to a nucleic acid, ora protein sequence, that links at least two heterologouspolynucleotides, or two heterologous polypeptides, into a singlemacromolecule, or that re-arranges one or more elements of at least onenatural nucleic acid or protein sequence. For example, the term“recombinant” can refer to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques.

As used herein, a “synthetic nucleotide sequence” or “syntheticpolynucleotide sequence” is a nucleotide sequence that is not known tooccur in nature or that is not naturally occurring. Generally, such asynthetic nucleotide sequence will comprise at least one nucleotidedifference when compared to any other naturally occurring nucleotidesequence.

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides, or analogs thereof. This term refers to theprimary structure of the molecule, and thus includes double- andsingle-stranded DNA, as well as double- and single-stranded RNA. It alsoincludes modified nucleic acids such as methylated and/or capped nucleicacids, nucleic acids containing modified bases, backbone modifications,and the like. The terms “nucleic acid” and “nucleotide sequence” areused interchangeably.

As used herein, the term “gene” refers to any segment of DNA associatedwith a biological function. Thus, genes include, but are not limited to,coding sequences and/or the regulatory sequences required for theirexpression. Genes can also include non-expressed DNA segments that, forexample, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

As used herein, the term “homologous” or “homologue” or “ortholog” isknown in the art and refers to related sequences that share a commonancestor or family member and are determined based on the degree ofsequence identity. The terms “homology,” “homologous,” “substantiallysimilar” and “corresponding substantially” are used interchangeablyherein. They refer to nucleic acid fragments wherein changes in one ormore nucleotide bases do not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant disclosure such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the disclosure encompasses more than the specificexemplary sequences. These terms describe the relationship between agene found in one species, subspecies, variety, cultivar or strain andthe corresponding or equivalent gene in another species, subspecies,variety, cultivar or strain. For purposes of this disclosure homologoussequences are compared. “Homologous sequences” or “homologues” or“orthologs” are thought, believed, or known to be functionally related.A functional relationship may be indicated in any one of a number ofways, including, but not limited to: (a) degree of sequence identityand/or (b) the same or similar biological function. Preferably, both (a)and (b) are indicated. Homology can be determined using softwareprograms readily available in the art, such as those discussed inCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30, section 7.718, Table 7.71. Some alignment programsare MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus(Scientific and Educational Software, Pennsylvania) and AlignX (VectorNTI, Invitrogen, Carlsbad, Calif.). Another alignment program isSequencher (Gene Codes, Ann Arbor, Mich.), using default parameters.

As used herein, the term “nucleotide change” refers to, e.g., nucleotidesubstitution, deletion, and/or insertion, as is well understood in theart. For example, mutations contain alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded protein or how the proteins are made.

As used herein, the term “protein modification” refers to, e.g., aminoacid substitution, amino acid modification, deletion, and/or insertion,as is well understood in the art.

As used herein, the term “at least a portion” or “fragment” of a nucleicacid or polypeptide means a portion having the minimal sizecharacteristics of such sequences, or any larger fragment of the fulllength molecule, up to and including the full length molecule. Afragment of a polynucleotide of the disclosure may encode a biologicallyactive portion of a genetic regulatory element. A biologically activeportion of a genetic regulatory element can be prepared by isolating aportion of one of the polynucleotides of the disclosure that comprisesthe genetic regulatory element and assessing activity as describedherein. Similarly, a portion of a polypeptide may be 4 amino acids, 5amino acids, 6 amino acids, 7 amino acids, and so on, going up to thefull length polypeptide. The length of the portion to be used willdepend on the particular application. A portion of a nucleic acid usefulas a hybridization probe may be as short as 12 nucleotides; in someembodiments, it is 20 nucleotides. A portion of a polypeptide useful asan epitope may be as short as 4 amino acids. A portion of a polypeptidethat performs the function of the full-length polypeptide wouldgenerally be longer than 4 amino acids.

Variant polynucleotides also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. Strategiesfor such DNA shuffling are known in the art. See, for example, Stemmer(1994) PNAS 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameriet al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.Biol. 272:336-347; Zhang et al. (1997) PNAS 94:4504-4509; Crameri etal.(1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and5,837,458. For PCR amplifications of the polynucleotides disclosedherein, oligonucleotide primers can be designed for use in PCR reactionsto amplify corresponding DNA sequences from cDNA or genomic DNAextracted from any organism of interest. Methods for designing PCRprimers and PCR cloning are generally known in the art and are disclosedin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nded., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See alsoInnis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach, thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product is induced, i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH. The (amplification) primer is preferably singlestranded for maximum efficiency in amplification. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact lengths of the primers will depend on manyfactors, including temperature and composition (A/T vs. G/C content) ofprimer. A pair of bi-directional primers consists of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

The terms “stringency” or “stringent hybridization conditions” refer tohybridization conditions that affect the stability of hybrids, e.g.,temperature, salt concentration, pH, formamide concentration and thelike. These conditions are empirically optimized to maximize specificbinding and minimize non-specific binding of primer or probe to itstarget nucleic acid sequence. The terms as used include reference toconditions under which a probe or primer will hybridize to its targetsequence, to a detectably greater degree than other sequences (e.g. atleast 2-fold over background). Stringent conditions are sequencedependent and will be different in different circumstances. Longersequences hybridize specifically at higher temperatures. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of a complementary target sequence hybridizes to aperfectly matched probe or primer. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 M Na+ion, typically about 0.01 to 1.0 M Na+ ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes or primers (e.g. 10 to 50 nucleotides) and at least about60° C. for long probes or primers (e.g. greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. Exemplary low stringentconditions or “conditions of reduced stringency” include hybridizationwith a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C. anda wash in 2×SSC at 40° C. Exemplary high stringency conditions includehybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in0.1×SSC at 60° C. Hybridization procedures are well known in the art andare described by e.g. Ausubel et al., 1998 and Sambrook et al., 2001. Insome embodiments, stringent conditions are hybridization in 0.25 MNa2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, 0.5-20% sodium dodecylsulfate at 45° C., such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, followed by awash in 5×SSC, containing 0.1% (w/v) sodium dodecyl sulfate, at 55° C.to 65° C.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Thepromoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity, and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue specificity of apromoter. Promoters may be derived in their entirety from a native gene,or be composed of different elements derived from different promotersfound in nature, or even comprise synthetic DNA segments. It isunderstood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of some variation may have identicalpromoter activity.

As used herein, a “constitutive promoter” is a promoter which is activeunder most conditions and/or during most development stages. There areseveral advantages to using constitutive promoters in expression vectorsused in biotechnology, such as: high level of production of proteinsused to select transgenic cells or organisms; high level of expressionof reporter proteins or scorable markers, allowing easy detection andquantification; high level of production of a transcription factor thatis part of a regulatory transcription system; production of compoundsthat requires ubiquitous activity in the organism; and production ofcompounds that are required during all stages of development.Non-limiting exemplary constitutive promoters include, CaMV 35Spromoter, opine promoters, ubiquitin promoter, alcohol dehydrogenasepromoter, etc.

As used herein, a “non-constitutive promoter” is a promoter which isactive under certain conditions, in certain types of cells, and/orduring certain development stages. For example, tissue specific, tissuepreferred, cell type specific, cell type preferred, inducible promoters,and promoters under development control are non-constitutive promoters.Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues.

As used herein, “inducible” or “repressible” promoter is a promoterwhich is under chemical or environmental factors control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions, certain chemicals, the presenceof light, acidic or basic conditions, etc.

As used herein, a “tissue specific” promoter is a promoter thatinitiates transcription only in certain tissues. Unlike constitutiveexpression of genes, tissue-specific expression is the result of severalinteracting levels of gene regulation. As such, in the art sometimes itis preferable to use promoters from homologous or closely relatedspecies to achieve efficient and reliable expression of transgenes inparticular tissues. This is one of the main reasons for the large amountof tissue-specific promoters isolated from particular tissues found inboth scientific and patent literature.

As used herein, the term “operably linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is regulated by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of regulatingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of thedisclosure can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

As used herein, the phrases “recombinant construct”, “expressionconstruct”, “chimeric construct”, “construct”, and “recombinant DNAconstruct” are used interchangeably herein. A recombinant constructcomprises an artificial combination of nucleic acid fragments, e.g.,regulatory and coding sequences that are not found together in nature.For example, a chimeric construct may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such constructmay be used by itself or may be used in conjunction with a vector. If avector is used then the choice of vector is dependent upon the methodthat will be used to transform host cells as is well known to thoseskilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the disclosure. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others. Vectors can be plasmids, viruses,bacteriophages, pro-viruses, phagemids, transposons, artificialchromosomes, and the like, that replicate autonomously or can integrateinto a chromosome of a host cell. A vector can also be a naked RNApolynucleotide, a naked DNA polynucleotide, a polynucleotide composed ofboth DNA and RNA within the same strand, a poly-lysine-conjugated DNA orRNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or thelike, that is not autonomously replicating. As used herein, the term“expression” refers to the production of a functional end-product e.g.,an mRNA or a protein (precursor or mature).

In some embodiments, the cell or organism has at least one heterologoustrait. As used herein, the term “heterologous trait” refers to aphenotype imparted to a transformed host cell or transgenic organism byan exogenous DNA segment, heterologous polynucleotide or heterologousnucleic acid. Various changes in phenotype are of interest to thepresent disclosure, including but not limited to modifying the fattyacid composition in milk, altering the carbohydrate content of milk,increasing an ungulate's yield of an economically important trait (e.g.,milk, milk fat, milk proteins, etc.) and the like. These results can beachieved by providing expression of heterologous products or increasedexpression of endogenous products in organisms using the methods andcompositions of the present disclosure.

As used herein, the term “MIC” means maximal information coefficient.MIC is a type of nonparamentric network analysis that identifies a score(MIC score) between active microbial strains of the present disclosureand at least one measured metadata (e.g., milk fat). Further, U.S.application Ser. No. 15/217,575, filed on Jul. 22, 2016 (issued as U.S.Pat. No. 9,540,676 on Jan. 10, 2017) is hereby incorporated by referencein its entirety.

The maximal information coefficient (MIC) is then calculated betweenstrains and metadata 3021 a, and between strains 3021 b; as seen in FIG.17. Results are pooled to create a list of all relationships and theircorresponding MIC scores 3022. If the relationship scores below a giventhreshold 3023, the relationship is deemed/identified as irrelevant 3023b. If the relationship is above a given threshold 3023, the relationshipdeemed/identified as relevant 2023 a, and is further subject to networkanalysis 3024. The following code fragment shows an exemplarymethodology for such analysis, according to one embodiment:

Read total list of relationships file as links threshold = 0.8 for i inrange(len(links)): if links >= threshold multiplier[i] = 1 elsemultiplier[i] = 0 end if links_temp = multiplier*links final_links =links_temp[links_temp != 0] savetxt(output_file,final_links)output_file.close( )

Based on the output of the network analysis, active strains are selected3025 for preparing products (e.g., ensembles, aggregates, and/or othersynthetic groupings) containing the selected strains. The output of thenetwork analysis can also be used to inform the selection of strains forfurther product composition testing.

The use of thresholds is discussed above for analyses anddeterminations. Thresholds can be, depending on the implementation andapplication: (1) empirically determined (e.g., based on distributionlevels, setting a cutoff at a number that removes a specified orsignificant portion of low level reads); (2) any non-zero value; (3)percentage/percentile based; (4) only strains whose normalized secondmarker (i.e., activity) reads is greater than normalized first marker(cell count) reads; (5) log2 fold change between activity and quantityor cell count; (6) normalized second marker (activity) reads is greaterthan mean second marker (activity) reads for entire sample (and/orsample set); and/or any magnitude threshold described above in additionto a statistical threshold (i.e., significance testing). The followingexample provides thresholding detail for distributions of RNA-basedsecond marker measurements with respect to DNA-based first markermeasurements, according to one embodiment.

As used herein “shelf-stable” refers to a functional attribute and newutility acquired by the microbes formulated according to the disclosure,which enable said microbes to exist in a useful/active state outside oftheir natural environment in the rumen (i.e. a markedly differentcharacteristic). Thus, shelf-stable is a functional attribute created bythe formulations/compositions of the disclosure and denoting that themicrobe formulated into a shelf-stable composition can exist outside therumen and under ambient conditions for a period of time that can bedetermined depending upon the particular formulation utilized, but ingeneral means that the microbes can be formulated to exist in acomposition that is stable under ambient conditions for at least a fewdays and generally at least one week. Accordingly, a “shelf-stableruminant supplement” is a composition comprising one or more microbes ofthe disclosure, said microbes formulated in a composition, such that thecomposition is stable under ambient conditions for at least one week,meaning that the microbes comprised in the composition (e.g. whole cell,spore, or lysed cell) are able to impart one or more beneficialphenotypic properties to a ruminant when administered (e.g. increasedmilk yield, improved milk compositional characteristics, improved rumenhealth, and/or modulation of the rumen microbiome).

Isolated Microbes

In some aspects, the present disclosure provides isolated microbes,including novel strains of microbes, presented in Table 1 and Table 3.

In other aspects, the present disclosure provides isolated wholemicrobial cultures of the microbes identified in Table 1 and Table 3.These cultures may comprise microbes at various concentrations.

In some aspects, the disclosure provides for utilizing one or moremicrobes selected from Table 1 and Table 3 to increase a phenotypictrait of interest in a ruminant.

In some embodiments, the disclosure provides isolated microbial speciesbelonging to taxonomic families of Clostridiaceae, Ruminococcaceae,Lachnospiraceae, Acidaminococcaceae, Peptococcaceae, Porphyromonadaceae,Prevotellaceae, Neocallimastigaceae, Saccharomycetaceae,Phaeosphaeriaceae, Erysipelotrichia, Anaerolinaeceae, Atopobiaceae,Botryosphaeriaceae, Eubacteriaceae, Acholeplasmataceae,Succinivibrionaceae, Lactobacillaceae, Selenomonadaceae,Burkholderiaceae, and Streptococcaceae.

In further embodiments, isolated microbial species may be selected fromgenera of family Clostridiaceae, including Acetanaerobacterium,Acetivibrio, Acidaminobacter, Alkahphilus, Anaerobacter, Anaerostipes,Anaerotruncus, Anoxynatronum, Bryantella, Butyricicoccus,Caldanaerocella, Caloramator, Caloranaerobacter, Caminicella, CandidatusArthromitus, Clostridium, Coprobacillus, Dorea, Ethanologenbacterium,Faecalibacterium, Garciella, Guggenheimella, Hespellia, Linmingia,Natronincola, Oxobacter, Parasporobacterium, Sarcina, Soehngenia,Sporobacter, Subdoligranulum, Tepidibacter, Tepidimicrobium,Thermobrachium, Thermohalobacter, and Tindallia.

In further embodiments, isolated microbial species may be selected fromgenera of family Ruminococcaceae, including Ruminococcus, Acetivibrio,Sporobacter, Anaerofilium, Papillibacter, Oscillospira, Gemmiger,Faecalibacterium, Fastidiosipila, Anaerotruncus, Ethanolingenens,Acetanaerobacterium, Subdoligranulum, Hydrogenoanaerobacterium, andCandidadus Soleaferrea.

In further embodiments, isolated microbial species may be selected fromgenera of family Lachnospiraceae, including Butyrivibrio, Roseburia,Lachnospira, Acetitomaculum, Coprococcus, Johnsonella, Catonella,Pseudobutyrivibrio, Syntrophococcus, Sporobacterium, Parasporobacterium,Lachnobacterium, Shuttleworthia, Dorea, Anaerostipes, Hespellia,Marvinbryantia, Oribacterium, Moryella, Blautia, Robinsoniella,Cellulosilyticum, Lachnoanaerobaculum, Stomatobaculum, Fusicatenibacter,Acetatifactor, and Eisenbergiella.

In further embodiments, isolated microbial species may be selected fromgenera of family Acidaminococcaceae, including Acidaminococcus,Phascolarctobacterium, Succiniclasticum, and Succinispira.

In further embodiments, isolated microbial species may be selected fromgenera of family Peptococcaceae, including Desulfotomaculum,Peptococcus, Desulfitobacterium, Syntrophobotulus, Dehalobacter,Sporotomaculum, Desulfosporosinus, Desulfonispora, Pelotomaculum,Thermincola, Cryptanaerobacter, Desulfitibacter, CandidatusDesulforudis, Desulfurispora, and Desulfitospora.

In further embodiments, isolated microbial species may be selected fromgenera of family Porphyromonadaceae, including Porphyromonas,Dysgonomonas, Tannerella, Odoribacter, Proteimphilum, Petrimonas,Paludibacter, Parabacteroides, Barnesiella, Candidatus Vestibaculum,Butyricimonas, Macellibacteroides, and Coprobacter.

In further embodiments, isolated microbial species may be selected fromgenera of family Anaerolinaeceae including Anaerolinea, Bellilinea,Leptolinea, Levilinea, Longilinea, Ornatilinea, and Pelolinea.

In further embodiments, isolated microbial species may be selected fromgenera of family Atopobiaceae including Atopbium and Olsenella.

In further embodiments, isolated microbial species may be selected fromgenera of family Eubacteriaceae including Acetobacterium, Alkalibacter,Alkalibaculum, Aminicella, Anaerofustis, Eubacterium, Gardella, andPseudoramibacter.

In further embodiments, isolated microbial species may be selected fromgenera of family Acholeplasmataceae including Acholeplasma.

In further embodiments, isolated microbial species may be selected fromgenera of family Succinivibrionaceae including Anaerobiospirillum,Ruminobacter, Succinatimonas, Succinimonas, and Succinivibrio.

In further embodiments, isolated microbial species may be selected fromgenera of family Lactobacillaceae including Lactobacillus,Paralactobacillus, Pediococcus, and Sharpea.

In further embodiments, isolated microbial species may be selected fromgenera of family Selenomonadaceae including Anaerovibrio, Centipeda,Megamonas, Mitsuokella, Pectinatus, Propionispira, Schwartzia,Selenomonas, and Zymophilus.

In further embodiments, isolated microbial species may be selected fromgenera of family Burkholderiaceae including Burkholderia, Chitinimonas,Cupriavidus, Lautropia, Limnobacter, Pandoraea, Paraburkholderia,Paucimonas, Polynucleobacter, Ralstonia, Thermothrix, and Wautersia.

In further embodiments, isolated microbial species may be selected fromgenera of family Streptococcaceae including Lactococcus, Lactovum, andStreptococcus.

In further embodiments, isolated microbial species may be selected fromgenera of family Anaerolinaeceae including Aestuariimicrobium, Arachnia,Auraticoccus, Brooklawnia, Friedmanniella, Granulicoccus, Luteococcus,Mariniluteicoccus, Microlunatus, Micropruina, Naumannella,Propionibacterium, Propionicicella, Propioniciclava, Propioniferax,Propionimicrobium, and Tessaracoccus.

In further embodiments, isolated microbial species may be selected fromgenera of family Prevotellaceae, including Paraprevotella, Prevotella,hallella, Xylanibacter, and Alloprevotella.

In further embodiments, isolated microbial species may be selected fromgenera of family Neocallimastigaceae, including Anaeromyces, Caecomyces,Cyllamyces, Neocallimastix, Orpinomyces, and Piromyces.

In further embodiments, isolated microbial species may be selected fromgenera of family Saccharomycetaceae, including Brettanomyces, Candida,Citeromyces, Cyniclomyces, Debaryomyces, Issatchenkia, Kazachstania(syn. Arxiozyma), Kluyveromyces, Komagataella, Kuraishia, Lachancea,Lodderomyces, Nakaseomyces, Pachysolen, Pichia, Saccharomyces,Spathaspora, Tetrapisispora, Vanderwaltozyma, Torulaspora, Williopsis,Zygosaccharomyces, and Zygotorulaspora.

In further embodiments, isolated microbial species may be selected fromgenera of family Erysipelotrichaceae, including Erysipelothrix,Solobacterium, Turicibacter, Faecalibaculum, Faecalicoccus,Faecalitalea, Holdemanella, Holdemania, Dielma, Eggerthia,Erysipelatoclostridium, Allobacterium, Breznakia, Bulleidia,Catenibacterium, Catenisphaera, and Coprobacillus.

In further embodiments, isolated microbial species may be selected fromgenera of family Phaeosphaeriaceae, including Barria, Bricookea,Carinispora, Chaetoplea, Eudarluca, Hadrospora, Isthmosporella,Katumotoa, Lautitia, Metameris, Mixtura, Neophaeosphaeria,Nodulosphaeria, Ophiosphaerella, Phaeosphaeris, Phaeosphaeriopsis,Setomelanomma, Stagonospora, Teratosphaeria, and Wilmia.

In further embodiments, isolated microbial species may be selected fromgenera of family Botryosphaeriaceae, including Amarenomyces,Aplosporella, Auerswaldiella, Botryosphaeria, Dichomera, Diplodia,Discochora, Dothidothia, Dothiorella, Fusicoccum, Granulodiplodia,Guignardia, Lasiodiplodia, Leptodothiorella, Leptodothiorella,Leptoguignardia, Macrophoma, Macrophomina, Nattrassia, Neodeightonia,Neofusicocum, Neoscytalidium, Otthia, Phaeobotryosphaeria,Phomatosphaeropsis, Phyllosticta, Pseudofusicoccum, Saccharata,Sivanesania, and Thyrostroma.

In some embodiments, the disclosure provides isolated microbial speciesbelonging to genera of: Clostridium, Ruminococcus, Roseburia,Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter,Pelotomaculum, Butyricicoccus, Tannerella, Prevotella, Butyricimonas,Piromyces, Candida, Vrystaatia, Orpinomyces, Neocallimastix, andPhyllosticta. In further embodiments, the disclosure provides isolatedmicrobial species belonging to the family of Lachnospiraceae, and theorder of Saccharomycetales. In further embodiments, the disclosureprovides isolated microbial species of Candida xylopsoci, Vrystaatiaaloeicola, and Phyllosticta capitalensis.

In some embodiments, a microbe from the taxa disclosed herein areutilized to impart one or more beneficial properties or improved traitsto milk in ruminants.

In some embodiments, the disclosure provides isolated microbial species,selected from the group consisting of: Clostridium, Ruminococcus,Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter,Pelotomaculum, Butyricicoccus, Tannerella, Prevotella, Butyricimonas,Piromyces, Pichia, Candida, Vrystaatia, Orpinomyces, Neocallimastix, andPhyllosticta.

In some embodiments, the disclosure provides novel isolated microbialstrains of species, selected from the group consisting of: Clostridium,Ruminococcus, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans,Papillibacter, Pelotomaculum, Butyricicoccus, Tannerella, Prevotella,Butyricimonas, Piromyces, Pichia, Candida, Vrystaatia, Orpinomyces,Neocallimastix, and Phyllosticta. Particular novel strains of theseaforementioned taxonomic groups can be found in Table 1 and/or Table 3.

Furthermore, the disclosure relates to microbes having characteristicssubstantially similar to that of a microbe identified in Table 1 orTable 3.

The isolated microbial species, and novel strains of said species,identified in the present disclosure, are able to impart beneficialproperties or traits to ruminant milk production.

For instance, the isolated microbes described in Table 1 and Table 3, orconsortia of said microbes, are able to increase total milk fat inruminant milk. The increase can be quantitatively measured, for example,by measuring the effect that said microbial application has upon themodulation of total milk fat.

In some embodiments, the isolated microbial strains are microbes of thepresent disclosure that have been genetically modified. In someembodiments, the genetically modified or recombinant microbes comprisepolynucleotide sequences which do not naturally occur in said microbes.In some embodiments, the microbes may comprise heterologouspolynucleotides. In further embodiments, the heterologouspolynucleotides may be operably linked to one or more polynucleotidesnative to the microbes.

In some embodiments, the heterologous polynucleotides may be reportergenes or selectable markers. In some embodiments, reporter genes may beselected from any of the family of fluorescence proteins (e.g., GFP,RFP, YFP, and the like), β-galactosidase, luciferase. In someembodiments, selectable markers may be selected from neomycinphosphotransferase, hygromycin phosphotransferase, aminoglycosideadenyltransferase, dihydrofolate reductase, acetolactase synthase,bromoxynil nitrilase, β-glucuronidase, dihydrogolate reductase, andchloramphenicol acetyltransferase. In some embodiments, the heterologouspolynucleotide may be operably linked to one or more promoter.

TABLE 4 Taxa (largely Genera) of the present disclosure not known tohave been utilized in animal agriculture. Intestinimonas AnaerolineaPseudobutyrivibrio Olsenella Eubacterium Catenisphaera FaecalibacteriumSolobacterium Blautia Ralsonia Coprococcus Casaltella AnaeroplasmaAcholeplasma Aminiphilus Mitsuokella Alistipes Sharpea OscillibacterNeocallimastix Odoribacter Pichia Tannerella CandidaHydrogenoanaerobacterium Orpinomyces Succinivibrio SugiyamaellaRuminobacter Cyllamyces Lachnospira Caecomyces SinimarinibacteriumTremella Hydrogenoanaerobacterium Turicibacter Clostridium XIVaAnaerolinea Saccharofermentans Piromyces Butyricicoccus OlsenellaPapillibacter Clostridium XICa Pelotomaculum ErysipelotrichaceaeLachnospiracea Solobacterium Anaeroplasma Ralstonia ClostridiumEubacterium Rikenella Lachnobacterium Tannerella Acholeplasma HowardellaSelenomonas Butyricimonas Sharpea Succinivibrio PhyllostictaRuminobacter Candida xylopsoc Syntrophococcus Candida apicolPseudobutyrivibrio Saccharomycetales Ascomycota Candida rugos

Microbial Consortia

In some aspects, the disclosure provides microbial consortia comprisinga combination of at least any two microbes selected from amongst themicrobes identified in Table 1 and/or Table 3.

In certain embodiments, the consortia of the present disclosure comprisetwo microbes, or three microbes, or four microbes, or five microbes, orsix microbes, or seven microbes, or eight microbes, or nine microbes, orten or more microbes. Said microbes of the consortia are differentmicrobial species, or different strains of a microbial species.

In some embodiments, the disclosure provides consortia, comprising: atleast two isolated microbial species belonging to genera of:Clostridium, Ruminococcus, Roseburia, Hydrogenoanaerobacterium,Saccharofermentans, Papillibacter, Pelotomaculum, Butyricicoccus,Tannerella, Prevotella, Butyricimonas, Piromyces, Pichia, Candida,Vrystaatia, Orpinomyces, Neocallimastix, and Phyllosticta. Particularnovel strains of species of these aforementioned genera can be found inTable 1 and/or Table 3.

In some embodiments, the disclosure provides consortia, comprising: atleast two isolated microbial species, selected from the group consistingof species of the family of Lachnospiraceae, and the order ofSaccharomycetales.

In particular aspects, the disclosure provides microbial consortia,comprising species as grouped in Tables 5-11. With respect to Tables5-11, the letters A through I represent a non-limiting selection ofmicrobes of the present disclosure, defined as:

A=Strain designation Ascusb_7 identified in Table 1;

B=Strain designation Ascusb_3138 identified in Table 1;

C=Strain designation Ascusb_82 identified in Table 1;

D=Strain designation Ascusb_119 identified in Table 1;

E=Strain designation Ascusb_1801 identified in Table 1;

F=Strain designation Ascusf_23 identified in Table 1;

G=Strain designation Ascusf_24 identified in Table 1;

H=Strain designation Ascusf_45 identified in Table 1; and

I=Strain designation Ascusf_15 identified in Table 1.

TABLE 5 Eight and Nine Strain Consortia A, B, C, D, E, F, G, H A, B, C,D, E, F, G, I A, B, C, D, E, F, H, I A, B, C, D, E, G, H, I A, B, C, D,F, G, H, I A, B, C, E, F, G, H, I A, B, D, E, F, G, H, I A, C, D, E, F,G, H, I B, C, D, E, F, G, H, I A, B, C, D, E, F, G, H, I

TABLE 6 Seven Strain Consortia A, B, C, D, E, F, G A, B, C, D, E, F, HA, B, C, D, E, F, I A, B, C, D, E, G, H A, B, C, D, E, G, I A, B, C, D,E, H, I A, B, C, D, F, G, H A, B, C, D, F, G, I A, B, C, D, F, H, I A,B, C, D, G, H, I A, B, C, E, F, G, H A, B, C, E, F, G, I A, B, C, E, F,H, I A, B, C, E, G, H, I A, B, C, F, G, H, I A, B, D, E, F, G, H A, B,D, E, F, G, I A, B, D, E, F, H, I A, B, D, E, G, H, I A, B, D, F, G, H,I A, B, E, F, G, H, I A, C, D, E, F, G, H A, C, D, E, F, G, I A, C, D,E, F, H, I A, C, D, E, G, H, I A, C, D, F, G, H, I A, C, E, F, G, H, IA, D, E, F, G, H, I B, C, D, E, F, G, H B, C, D, E, F, G, I B, C, D, E,F, H, I B, C, D, E, G, H, I B, C, D, F, G, H, I B, C, E, F, G, H, I B,D, E, F, G, H, I C, D, E, F, G, H, I

TABLE 7 Six Strain Consortia A, B, C, D, E, F A, B, C, D, E, G A, B, C,D, E, H A, B, C, D, E, I A, B, C, D, F, G A, B, C, D, F, H A, B, C, D,F, I A, B, C, D, G, H A, B, C, D, G, I A, B, C, D, H, I A, B, C, E, F, GA, B, C, E, F, H A, B, C, E, F, I A, B, C, E, G, H A, B, C, E, G, I A,B, C, E, H, I A, B, C, F, G, H A, B, C, F, G, I A, B, C, F, H, I A, B,C, G, H, I A, B, D, E, F, G A, B, D, E, F, H A, B, D, E, F, I A, B, D,E, G, H A, B, D, E, G, I A, B, D, E, H, I A, B, D, F, G, H A, B, D, F,G, I D, E, F, G, H, I C, E, F, G, H, I A, B, D, F, H, I A, B, D, G, H, IA, B, E, F, G, H A, B, E, F, G, I A, B, E, F, H, I A, B, E, G, H, I A,B, F, G, H, I A, C, D, E, F, G A, C, D, E, F, H A, C, D, E, F, I A, C,D, E, G, H A, C, D, E, G, I A, C, D, E, H, I A, C, D, F, G, H A, C, D,F, G, I A, C, D, F, H, I A, C, D, G, H, I A, C, E, F, G, H A, C, E, F,G, I A, C, E, F, H, I A, C, E, G, H, I A, C, F, G, H, I A, D, E, F, G, HA, D, E, F, G, I A, D, E, F, H, I A, D, E, G, H, I A, D, F, G, H, I A,E, F, G, H, I B, C, D, E, F, G B, C, D, E, F, H B, C, D, E, F, I B, C,D, E, G, H B, C, D, E, G, I B, C, D, E, H, I B, C, D, F, G, H B, C, D,F, G, I B, C, D, F, H, I B, C, D, G, H, I B, C, E, F, G, H B, C, E, F,G, I B, C, E, F, H, I B, C, E, G, H, I B, C, F, G, H, I B, D, E, F, G, HB, D, E, F, G, I B, D, E, F, H, I B, D, E, G, H, I B, D, F, G, H, I B,E, F, G, H, I C, D, E, F, G, H C, D, E, F, G, I C, D, E, F, H, I C, D,E, G, H, I C, D, F, G, H, I

TABLE 8 Five Strain Consortia A, B, C, D, E A, B, C, D, F A, B, C, D, GA, B, C, D, H A, B, C, D, I A, B, C, E, F A, B, C, E, G A, B, C, E, H A,B, C, F, H A, B, C, F, G A, B, C, F, I A, B, C, G, H A, B, C, G, I A, B,C, H, I A, B, D, E, F A, B, D, E, G A, B, D, E, I A, B, D, F, G A, B, D,F, H A, B, D, F, I A, B, D, G, H A, B, D, G, I A, B, D, H, I A, B, E, F,G A, B, E, F, I A, B, E, G, H A, B, E, G, I A, B, E, H, I A, B, F, G, HA, B, F, G, I A, B, F, H, I A, B, G, H, I A, C, D, E, G A, C, D, E, H A,C, D, E, I A, C, D, F, G A, C, D, F, H A, C, D, F, I A, C, D, G, H A, C,D, G, I A, C, E, F, G A, C, E, F, H A, C, E, F, I A, C, E, G, H A, C, E,G, I A, C, E, H, I A, C, F, G, H A, C, F, G, I A, C, G, H, I A, D, E, F,G A, D, E, F, H A, D, E, F, I A, D, E, G, H A, D, E, G, I A, D, E, H, IA, D, F, G, H A, D, F, H, I A, D, G, H, I A, E, F, G, H A, E, F, G, I A,E, F, H, I A, E, G, H, I A, F, G, H, I B, C, D, E, F B, C, D, E, H B, C,D, E, I B, C, D, F, G B, C, D, F, H B, C, D, F, I B, C, D, G, H B, C, D,G, I B, C, D, H, I B, C, E, F, H B, C, E, F, I B, C, E, G, H B, C, E, G,I B, C, E, H, I B, C, F, G, H B, C, F, G, I B, C, F, H, I B, D, E, F, GB, D, E, F, H B, D, E, F, I B, D, E, G, H B, D, E, G, I B, D, E, H, I B,D, F, G, H B, D, F, G, I B, D, G, H, I B, E, F, G, H B, E, F, G, I B, E,F, H, I B, E, G, H, I B, F, G, H, I C, D, E, F, G C, D, E, F, H C, D, E,G, H C, D, E, G, I C, D, E, H, I C, D, F, G, H C, D, F, G, I C, D, F, H,I C, D, G, H, I C, E, F, G, H C, E, F, H, I C, E, G, H, I C, F, G, H, ID, E, F, G, H D, E, F, G, I D, E, F, H, I D, E, G, H, I D, F, G, H, I A,B, C, E, I A, B, D, E, H A, B, E, F, H A, C, D, E, F A, C, D, H, I A, C,F, H, I A, D, F, G, I B, C, D, E, G B, C, E, F, G B, C, G, H, I B, D, F,H, I C, D, E, F, I C, E, F, G, I E, F, G, H, I

TABLE 9 Four Strain Consortia A, B, C, D A, B, C, E A, B, C, F A, B, C,G A, B, C, H A, B, C, I A, B, D, E A, B, D, F D, G, H, I A, B, D, G A,B, D, H A, B, D, I A, B, E, F A, B, E, G A, B, E, H A, B, E, I A, B, F,G E, F, G, H A, B, F, H A, D, F, H A, D, F, I A, D, G, H A, D, G, I A,D, H, I A, E, F, G A, E, F, H E, F, G, I A, B, F, I A, B, G, H A, B, G,I A, B, H, I A, C, D, E A, C, D, F A, C, D, G A, C, D, H E, F, H, I A,C, D, I A, C, E, F A, C, E, G A, C, E, H A, C, E, I A, C, F, G A, C, F,H A, C, F, I E, G, H, I A, C, G, H A, C, G, I A, C, H, I A, D, E, F A,D, E, G A, D, E, H A, D, E, I A, D, F, G F, G, H, I A, E, F, I A, E, G,H A, E, G, I A, E, H, I A, F, G, H A, F, G, I A, F, H, I A, G, H, I D,E, F, H B, C, D, E B, C, D, F B, C, D, G B, C, D, H B, C, D, I B, C, E,F B, C, E, G B, C, E, H D, E, F, I B, C, E, I B, C, F, G B, C, F, H B,C, F, I B, C, G, H B, C, G, I B, C, H, I B, D, E, F D, E, G, H B, D, E,G B, D, E, H B, D, E, I B, D, F, G B, D, F, H B, D, F, I B, D, G, H B,D, G, I D, E, G, I B, D, H, I B, E, F, G B, E, F, H B, E, F, I B, E, G,H B, E, G, I B, E, H, I B, F, G, H D, E, H, I B, F, G, I B, F, H, I B,G, H, I C, D, E, F C, D, E, G C, D, E, H C, D, E, I C, D, F, G D, F, G,H C, D, F, H C, D, F, I C, D, G, H C, D, G, I C, D, H, I C, E, F, G C,E, F, H C, E, F, I D, F, G, I C, E, G, H C, E, G, I C, E, H, I C, F, G,H C, F, G, I C, F, H, I C, G, H, I D, E, F, G D, F, H, I

TABLE 10 Three Strain Consortia A, B, C A, B, D A, B, E A, B, F A, B, GA, B, H A, B, I A, C, D A, C, E G, H, I E, F, H A, C, F A, C, G A, C, HA, C, I A, D, E A, D, F A, D, G A, D, H A, D, I F, H, I E, F, G A, E, FA, E, G A, E, H A, E, I A, F, G A, F, H A, F, I A, G, H A, G, I F, G, ID, H, I A, H, I B, C, D B, C, E B, C, F B, C, G B, C, H B, C, I B, D, EB, D, F F, G, H D, G, I B, D, G B, D, H B, D, I B, E, F B, E, G B, E, HB, E, I B, F, G B, F, H E, H, I E, F, I B, F, I B, G, H B, G, I B, H, IC, D, E C, D, F C, D, G C, D, H C, D, I E, G, I D, G, H C, E, F C, E, GC, E, H C, E, I C, F, G C, F, H C, F, I C, G, H C, G, I E, G, H D, F, IC, H, I D, E, F D, E, G D, E, H D, E, I D, F, G D, F, H

TABLE 11 Two Strain Consortia A, B A, C A, D A, E A, F A, G A, H A, I B,C B, D B, E B, F B, G B, H B, I C, D C, E C, F C, G C, H C, I D, E D, FD, G D, H D, I E, F E, G E, H E, I F, G F, H F, I G, H G, I H, I

In some embodiments, the microbial consortia may be selected from anymember group from Tables 5-11.

Isolated Microbes—Source Material

The microbes of the present disclosure were obtained, among otherplaces, at various locales in the United States from thegastrointestinal tract of cows.

Isolated Microbes—Microbial Culture Techniques

The microbes of Table 1 and Table 3 were matched to their nearesttaxonomic groups by utilizing classification tools of the RibosomalDatabase Project (RDP) for 16s rRNA sequences and the User-friendlyNordic ITS Ectomycorrhiza (UNITE) database for ITS rRNA sequences.Examples of matching microbes to their nearest taxa may be found in Lanet al. (2012. PLOS one. 7(3):e32491), Schloss and Westcott (2011. Appl.Environ. Microbiol. 77(10):3219-3226), and Koljalg et al. (2005. NewPhytologist. 166(3): 1063-1068).

The isolation, identification, and culturing of the microbes of thepresent disclosure can be effected using standard microbiologicaltechniques. Examples of such techniques may be found in Gerhardt, P.(ed.) Methods for General and Molecular Microbiology. American Societyfor Microbiology, Washington, D.C. (1994) and Lennette, E. H. (ed.)Manual of Clinical Microbiology, Third Edition. American Society forMicrobiology, Washington, D.C. (1980), each of which is incorporated byreference.

Isolation can be effected by streaking the specimen on a solid medium(e.g., nutrient agar plates) to obtain a single colony, which ischaracterized by the phenotypic traits described hereinabove (e.g., Grampositive/negative, capable of forming spores aerobically/anaerobically,cellular morphology, carbon source metabolism, acid/base production,enzyme secretion, metabolic secretions, etc.) and to reduce thelikelihood of working with a culture which has become contaminated.

For example, for microbes of the disclosure, biologically pure isolatescan be obtained through repeated subculture of biological samples, eachsubculture followed by streaking onto solid media to obtain individualcolonies or colony forming units. Methods of preparing, thawing, andgrowing lyophilized bacteria are commonly known, for example, Gherna, R.L. and C. A. Reddy. 2007. Culture Preservation, p 1019-1033. In C. A.Reddy, T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt, andL. R. Snyder, eds. American Society for Microbiology, Washington, D.C.,1033 pages; herein incorporated by reference. Thus freeze dried liquidformulations and cultures stored long term at −70° C. in solutionscontaining glycerol are contemplated for use in providing formulationsof the present disclosure.

The microbes of the disclosure can be propagated in a liquid mediumunder aerobic conditions, or alternatively anaerobic conditions. Mediumfor growing the bacterial strains of the present disclosure includes acarbon source, a nitrogen source, and inorganic salts, as well asspecially required substances such as vitamins, amino acids, nucleicacids and the like. Examples of suitable carbon sources which can beused for growing the microbes include, but are not limited to, starch,peptone, yeast extract, amino acids, sugars such as glucose, arabinose,mannose, glucosamine, maltose, and the like; salts of organic acids suchas acetic acid, fumaric acid, adipic acid, propionic acid, citric acid,gluconic acid, malic acid, pyruvic acid, malonic acid and the like;alcohols such as ethanol and glycerol and the like; oil or fat such assoybean oil, rice bran oil, olive oil, corn oil, sesame oil. The amountof the carbon source added varies according to the kind of carbon sourceand is typically between 1 to 100 gram(s) per liter of medium.Preferably, glucose, starch, and/or peptone is contained in the mediumas a major carbon source, at a concentration of 0.1-5% (W/V). Examplesof suitable nitrogen sources which can be used for growing the bacterialstrains of the present disclosure include, but are not limited to, aminoacids, yeast extract, tryptone, beef extract, peptone, potassiumnitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammoniumphosphate, ammonia or combinations thereof. The amount of nitrogensource varies according to the type of nitrogen source, typicallybetween 0.1 to 30 gram per liter of medium. The inorganic salts,potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodiumhydrogen phosphate, magnesium sulfate, magnesium chloride, ferricsulfate, ferrous sulfate, ferric chloride, ferrous chloride, manganoussulfate, manganous chloride, zinc sulfate, zinc chloride, cupricsulfate, calcium chloride, sodium chloride, calcium carbonate, sodiumcarbonate can be used alone or in combination. The amount of inorganicacid varies according to the kind of the inorganic salt, typicallybetween 0.001 to 10 gram per liter of medium. Examples of speciallyrequired substances include, but are not limited to, vitamins, nucleicacids, yeast extract, peptone, meat extract, malt extract, dried yeastand combinations thereof. Cultivation can be effected at a temperature,which allows the growth of the microbial strains, essentially, between20° C. and 46° C. In some aspects, a temperature range is 30° C.-39° C.For optimal growth, in some embodiments, the medium can be adjusted topH 6.0-7.4. It will be appreciated that commercially available media mayalso be used to culture the microbial strains, such as Nutrient Broth orNutrient Agar available from Difco, Detroit, Mich. It will beappreciated that cultivation time may differ depending on the type ofculture medium used and the concentration of sugar as a major carbonsource.

In some aspects, cultivation lasts between 24-96 hours. Microbial cellsthus obtained are isolated using methods, which are well known in theart. Examples include, but are not limited to, membrane filtration andcentrifugal separation. The pH may be adjusted using sodium hydroxideand the like and the culture may be dried using a freeze dryer, untilthe water content becomes equal to 4% or less. Microbial co-cultures maybe obtained by propagating each strain as described hereinabove. In someaspects, microbial multi-strain cultures may be obtained by propagatingtwo or more of the strains described hereinabove. It will be appreciatedthat the microbial strains may be cultured together when compatibleculture conditions can be employed.

Isolated Microbes—Microbial Strains

Microbes can be distinguished into a genus based on polyphasic taxonomy,which incorporates all available phenotypic and genotypic data into aconsensus classification (Vandamme et al. 1996. Polyphasic taxonomy, aconsensus approach to bacterial systematics. Microbial Rev 1996,60:407-438). One accepted genotypic method for defining species is basedon overall genomic relatedness, such that strains which shareapproximately 70% or more relatedness using DNA-DNA hybridization, with5° C. or less ΔT_(m) (the difference in the melting temperature betweenhomologous and heterologous hybrids), under standard conditions, areconsidered to be members of the same species. Thus, populations thatshare greater than the aforementioned 70% threshold can be considered tobe variants of the same species. Another accepted genotypic method fordefining species is to isolate marker genes of the present disclosure,sequence these genes, and align these sequenced genes from multipleisolates or variants. The microbes are interpreted as belonging to thesame species if one or more of the sequenced genes share at least 97%sequence identity.

The 16S or 18S rRNA sequences or ITS sequences are often used for makingdistinctions between species and strains, in that if one of theaforementioned sequences share less than a specified percent sequenceidentity from a reference sequence, then the two organisms from whichthe sequences were obtained are said to be of different species orstrains.

Thus, one could consider microbes to be of the same species, if theyshare at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identityacross the 16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence.

Further, one could define microbial strains of a species, as those thatshare at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identityacross the 16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence.

In one embodiment, microbial strains of the present disclosure includethose that comprise polynucleotide sequences that share at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any oneof SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 39, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 2045, 2046, 2047, 2048, 2049, 2050, 2051,2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063,2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075,2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087,2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099,2100, 2101, 2102, 2103, 2104, 2105, 2106, and 2107. In a furtherembodiment, microbial strains of the present disclosure include thosethat comprise polynucleotide sequences that share at least 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any one ofSEQ ID NOs:1-2107.

Comparisons may also be made with 23S rRNA sequences against referencesequences.

Unculturable microbes often cannot be assigned to a definite species inthe absence of a phenotype determination, the microbes can be given acandidatus designation within a genus provided their 16S or 18S rRNAsequences or ITS sequences subscribes to the principles of identity withknown species.

One approach is to observe the distribution of a large number of strainsof closely related species in sequence space and to identify clusters ofstrains that are well resolved from other clusters. This approach hasbeen developed by using the concatenated sequences of multiple core(house-keeping) genes to assess clustering patterns, and has been calledmultilocus sequence analysis (MLSA) or multilocus sequence phylogeneticanalysis. MLSA has been used successfully to explore clustering patternsamong large numbers of strains assigned to very closely related speciesby current taxonomic methods, to look at the relationships between smallnumbers of strains within a genus, or within a broader taxonomicgrouping, and to address specific taxonomic questions. More generally,the method can be used to ask whether bacterial species exist—that is,to observe whether large populations of similar strains invariably fallinto well-resolved clusters, or whether in some cases there is a geneticcontinuum in which clear separation into clusters is not observed.

In order to more accurately make a determination of genera, adetermination of phenotypic traits, such as morphological, biochemical,and physiological characteristics are made for comparison with areference genus archetype. The colony morphology can include color,shape, pigmentation, production of slime, etc. Features of the cell aredescribed as to shape, size, Gram reaction, extracellular material,presence of endospores, flagella presence and location, motility, andinclusion bodies. Biochemical and physiological features describe growthof the organism at different ranges of temperature, pH, salinity andatmospheric conditions, growth in presence of different sole carbon andnitrogen sources. One of ordinary skill in the art would be reasonablyapprised as to the phenotypic traits that define the genera of thepresent disclosure.

In one embodiment, the microbes taught herein were identified utilizing16S rRNA gene sequences and ITS sequences. It is known in the art that16S rRNA contains hypervariable regions that can providespecies/strain-specific signature sequences useful for bacterialidentification, and that ITS sequences can also providespecies/strain-specific signature sequences useful for fungalidentification.

Phylogenetic analysis using the rRNA genes and/or ITS sequences are usedto define “substantially similar” species belonging to common genera andalso to define “substantially similar” strains of a given taxonomicspecies. Furthermore, physiological and/or biochemical properties of theisolates can be utilized to highlight both minor and significantdifferences between strains that could lead to advantageous behavior inruminants.

Compositions of the present disclosure may include combinations offungal spores and bacterial spores, fungal spores and bacterialvegetative cells, fungal vegetative cells and bacterial spores, fungalvegetative cells and bacterial vegetative cells. In some embodiments,compositions of the present disclosure comprise bacteria only in theform of spores. In some embodiments, compositions of the presentdisclosure comprise bacteria only in the form of vegetative cells. Insome embodiments, compositions of the present disclosure comprisebacteria in the absence of fungi. In some embodiments, compositions ofthe present disclosure comprise fungi in the absence of bacteria.

Bacterial spores may include endospores and akinetes. Fungal spores mayinclude statismospores, ballistospores, autospores, aplanospores,zoospores, mitospores, megaspores, microspores, meiospores,chlamydospores, urediniospores, teliospores, oospores, carpospores,tetraspores, sporangiospores, zygospores, ascospores, basidiospores,ascospores, and asciospores.

In some embodiments, spores of the composition germinate uponadministration to animals of the present disclosure. In someembodiments, spores of the composition germinate only uponadministration to animals of the present disclosure.

Microbial Compositions

In some embodiments, the microbes of the disclosure are combined intomicrobial compositions.

In some embodiments, the microbial compositions include ruminant feed,such as cereals (barley, maize, oats, and the like); starches (tapiocaand the like); oilseed cakes; and vegetable wastes. In some embodiments,the microbial compositions include vitamins, minerals, trace elements,emulsifiers, aromatizing products, binders, colorants, odorants,thickening agents, and the like.

In some embodiments, the microbial compositions of the presentdisclosure are solid. Where solid compositions are used, it may bedesired to include one or more carrier materials including, but notlimited to: mineral earths such as silicas, talc, kaolin, limestone,chalk, clay, dolomite, diatomaceous earth; calcium sulfate; magnesiumsulfate; magnesium oxide; products of vegetable origin such as cerealmeals, tree bark meal, wood meal, and nutshell meal.

In some embodiments, the microbial compositions of the presentdisclosure are liquid. In further embodiments, the liquid comprises asolvent that may include water or an alcohol, and other animal-safesolvents. In some embodiments, the microbial compositions of the presentdisclosure include binders such as animal-safe polymers,carboxymethylcellulose, starch, polyvinyl alcohol, and the like.

In some embodiments, the microbial compositions of the presentdisclosure comprise thickening agents such as silica, clay, naturalextracts of seeds or seaweed, synthetic derivatives of cellulose, guargum, locust bean gum, alginates, and methylcelluloses. In someembodiments, the microbial compositions comprise anti-settling agentssuch as modified starches, polyvinyl alcohol, xanthan gum, and the like.

In some embodiments, the microbial compositions of the presentdisclosure comprise colorants including organic chromophores classifiedas nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine,anthraquinone, azine, diphenylmethane, indamine, indophenol, methine,oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene.In some embodiments, the microbial compositions of the presentdisclosure comprise trace nutrients such as salts of iron, manganese,boron, copper, cobalt, molybdenum and zinc.

In some embodiments, the microbial compositions of the presentdisclosure comprise an animal-safe virucide or nematicide.

In some embodiments, microbial compositions of the present disclosurecomprise saccharides (e.g., monosaccharides, disaccharides,trisaccharides, polysaccharides, oligosaccharides, and the like),polymeric saccharides, lipids, polymeric lipids, lipopolysaccharides,proteins, polymeric proteins, lipoproteins, nucleic acids, nucleic acidpolymers, silica, inorganic salts and combinations thereof. In a furtherembodiment, microbial compositions comprise polymers of agar, agarose,gelrite, gellan gumand the like. In some embodiments, microbialcompositions comprise plastic capsules, emulsions (e.g., water and oil),membranes, and artificial membranes. In some embodiments, emulsions orlinked polymer solutions may comprise microbial compositions of thepresent disclosure. See Harel and Bennett (U.S. Pat. No. 8,460,726B2).

In some embodiments, microbial compositions of the present disclosureoccur in a solid form (e.g., dispersed lyophilized spores) or a liquidform (microbes interspersed in a storage medium).

In some embodiments, microbial compositions of the present disclosurecomprise one or more preservatives. The preservatives may be in liquidor gas formulations. The preservatives may be selected from one or moreof monosaccharide, disaccharide, trisaccharide, polysaccharide, aceticacid, ascorbic acid, calcium ascorbate, erythorbic acid, iso-ascorbicacid, erythrobic acid, potassium nitrate, sodium ascorbate, sodiumerythorbate, sodium iso-ascorbate, sodium nitrate, sodium nitrite,nitrogen, benzoic acid, calcium sorbate, ethyl lauroyl arginate,methyl-p-hydroxy benzoate, methyl paraben, potassium acetate, potassiumbenzoiate, potassium bisulphite, potassium diacetate, potassium lactate,potassium metabisulphite, potassium sorbate, propyl-p-hydroxy benzoate,propyl paraben, sodium acetate, sodium benzoate, sodium bisulphite,sodium nitrite, sodium diacetate, sodium lactate, sodium metabisulphite,sodium salt of methyl-p-hydroxy benzoic acid, sodium salt ofpropyl-p-hydroxy benzoic acid, sodium sulphate, sodium sulfite, sodiumdithionite, sulphurous acid, calcium propionate, dimethyl dicarbonate,natamycin, potassium sorbate, potassium bisulfite, potassiummetabisulfite, propionic acid, sodium diacetate, sodium propionate,sodium sorbate, sorbic acid, ascorbic acid, ascorbyl palmitate, ascorbylstearate, butylated hydro-xyanisole, butylated hydroxytoluene (BHT),butylated hydroxyl anisole (BHA), citric acid, citric acid esters ofmono- and/or diglycerides, L-cysteine, L-cysteine hydrochloride, gumguaiacum, gum guaiac, lecithin, lecithin citrate, monoglyceride citrate,monoisopropyl citrate, propyl gallate, sodium metabisulphite, tartaricacid, tertiary butyl hydroquinone, stannous chloride, thiodipropionicacid, dilauryl thiodipropionate, distearyl thiodipropionate, ethoxyquin,sulfur dioxide, formic acid, or tocopherol(s).

In some embodiments, microbial compositions of the present disclosureinclude bacterial and/or fungal cells in spore form, vegetative cellform, and/or lysed cell form. In one embodiment, the lysed cell formacts as a mycotoxin binder, e.g. mycotoxins binding to dead cells.

In some embodiments, the microbial compositions are shelf stable in arefrigerator (35-40° F.) for a period of at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60days. In some embodiments, the microbial compositions are shelf stablein a refrigerator (35-40° F.) for a period of at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60weeks.

In some embodiments, the microbial compositions are shelf stable at roomtemperature (68-72° F.) or between 50-77° F. for a period of at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, or 60 days. In some embodiments, the microbial compositions areshelf stable at room temperature (68-72° F.) or between 50-77° F. foraperiod of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions are shelf stable at-23-35° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In someembodiments, the microbial compositions are shelf stable at −23-35° F.for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions are shelf stable at77-100° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In someembodiments, the microbial compositions are shelf stable at 77-100° F.for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions are shelf stable at101-213° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In someembodiments, the microbial compositions are shelf stable at 101-213° F.fora period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of about 1 to100, about 1 to 95, about 1 to 90, about 1 to 85, about 1 to 80, about 1to 75, about 1 to 70, about 1 to 65, about 1 to 60, about 1 to 55, about1 to 50, about 1 to 45, about 1 to 40, about 1 to 35, about 1 to 30,about 1 to 25, about 1 to 20, about 1 to 15, about 1 to 10, about 1 to5, about 5 to 100, about 5 to 95, about 5 to 90, about 5 to 85, about 5to 80, about 5 to 75, about 5 to 70, about 5 to 65, about 5 to 60, about5 to 55, about 5 to 50, about 5 to 45, about 5 to 40, about 5 to 35,about 5 to 30, about 5 to 25, about 5 to 20, about 5 to 15, about 5 to10, about 10 to 100, about 10 to 95, about 10 to 90, about 10 to 85,about 10 to 80, about 10 to 75, about 10 to 70, about 10 to 65, about 10to 60, about 10 to 55, about 10 to 50, about 10 to 45, about 10 to 40,about 10 to 35, about 10 to 30, about 10 to 25, about 10 to 20, about 10to 15, about 15 to 100, about 15 to 95, about 15 to 90, about 15 to 85,about 15 to 80, about 15 to 75, about 15 to 70, about 15 to 65, about 15to 60, about 15 to 55, about 15 to 50, about 15 to 45, about 15 to 40,about 15 to 35, about 15 to 30, about 15 to 25, about 15 to 20, about 20to 100, about 20 to 95, about 20 to 90, about 20 to 85, about 20 to 80,about 20 to 75, about 20 to 70, about 20 to 65, about 20 to 60, about 20to 55, about 20 to 50, about 20 to 45, about 20 to 40, about 20 to 35,about 20 to 30, about 20 to 25, about 25 to 100, about 25 to 95, about25 to 90, about 25 to 85, about 25 to 80, about 25 to 75, about 25 to70, about 25 to 65, about 25 to 60, about 25 to 55, about 25 to 50,about 25 to 45, about 25 to 40, about 25 to 35, about 25 to 30, about 30to 100, about 30 to 95, about 30 to 90, about 30 to 85, about 30 to 80,about 30 to 75, about 30 to 70, about 30 to 65, about 30 to 60, about 30to 55, about 30 to 50, about 30 to 45, about 30 to 40, about 30 to 35,about 35 to 100, about 35 to 95, about 35 to 90, about 35 to 85, about35 to 80, about 35 to 75, about 35 to 70, about 35 to 65, about 35 to60, about 35 to 55, about 35 to 50, about 35 to 45, about 35 to 40,about 40 to 100, about 40 to 95, about 40 to 90, about 40 to 85, about40 to 80, about 40 to 75, about 40 to 70, about 40 to 65, about 40 to60, about 40 to 55, about 40 to 50, about 40 to 45, about 45 to 100,about 45 to 95, about 45 to 90, about 45 to 85, about 45 to 80, about 45to 75, about 45 to 70, about 45 to 65, about 45 to 60, about 45 to 55,about 45 to 50, about 50 to 100, about 50 to 95, about 50 to 90, about50 to 85, about 50 to 80, about 50 to 75, about 50 to 70, about 50 to65, about 50 to 60, about 50 to 55, about 55 to 100, about 55 to 95,about 55 to 90, about 55 to 85, about 55 to 80, about 55 to 75, about 55to 70, about 55 to 65, about 55 to 60, about 60 to 100, about 60 to 95,about 60 to 90, about 60 to 85, about 60 to 80, about 60 to 75, about 60to 70, about 60 to 65, about 65 to 100, about 65 to 95, about 65 to 90,about 65 to 85, about 65 to 80, about 65 to 75, about 65 to 70, about 70to 100, about 70 to 95, about 70 to 90, about 70 to 85, about 70 to 80,about 70 to 75, about 75 to 100, about 75 to 95, about 75 to 90, about75 to 85, about 75 to 80, about 80 to 100, about 80 to 95, about 80 to90, about 80 to 85, about 85 to 100, about 85 to 95, about 85 to 90,about 90 to 100, about 90 to 95, or 95 to 100 weeks

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of 1 to 100, 1to 95, 1 to 90, 1 to 85, 1 to 80, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1to 55, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1to 15, 1 to 10, 1 to 5, 5 to 100, 5 to 95, 5 to 90, 5 to 85, 5 to 80, 5to 75, 5 to 70, 5 to 65, 5 to 60, 5 to 55, 5 to 50, 5 to 45, 5 to 40, 5to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 100, 10 to 95,10 to 90, 10 to 85, 10 to 80, 10 to 75, 10 to 70, 10 to 65, 10 to 60, 10to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to20, 10 to 15, 15 to 100, 15 to 95, 15 to 90, 15 to 85, 15 to 80, 15 to75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 100, 20 to 95, 20 to90, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60, 20 to55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to100, 25 to 95, 25 to 90, 25 to 85, 25 to 80, 25 to 75, 25 to 70, 25 to65, 25 to 60, 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to30, 30 to 100, 30 to 95, 30 to 90, 30 to 85, 30 to 80, 30 to 75, 30 to70, 30 to 65, 30 to 60, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to35, 35 to 100, 35 to 95, 35 to 90, 35 to 85, 35 to 80, 35 to 75, 35 to70, 35 to 65, 35 to 60, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to100, 40 to 95, 40 to 90, 40 to 85, 40 to 80, 40 to 75, 40 to 70, 40 to65, 40 to 60, 40 to 55, 40 to 50, 40 to 45, 45 to 100, 45 to 95, 45 to90, 45 to 85, 45 to 80, 45 to 75, 45 to 70, 45 to 65, 45 to 60, 45 to55, 45 to 50, 50 to 100, 50 to 95, 50 to 90, 50 to 85, 50 to 80, 50 to75, 50 to 70, 50 to 65, 50 to 60, 50 to 55, 55 to 100, 55 to 95, 55 to90, 55 to 85, 55 to 80, 55 to 75, 55 to 70, 55 to 65, 55 to 60, 60 to100, 60 to 95, 60 to 90, 60 to 85, 60 to 80, 60 to 75, 60 to 70, 60 to65, 65 to 100, 65 to 95, 65 to 90, 65 to 85, 65 to 80, 65 to 75, 65 to70, 70 to 100, 70 to 95, 70 to 90, 70 to 85, 70 to 80, 70 to 75, 75 to100, 75 to 95, 75 to 90, 75 to 85, 75 to 80, 80 to 100, 80 to 95, 80 to90, 80 to 85, 85 to 100, 85 to 95, 85 to 90, 90 to 100, 90 to 95, or 95to 100 weeks.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of about 1 to36, about 1 to 34, about 1 to 32, about 1 to 30, about 1 to 28, about 1to 26, about 1 to 24, about 1 to 22, about 1 to 20, about 1 to 18, about1 to 16, about 1 to 14, about 1 to 12, about 1 to 10, about 1 to 8,about 1 to 6, about 1 one 4, about 1 to 2, about 4 to 36, about 4 to 34,about 4 to 32, about 4 to 30, about 4 to 28, about 4 to 26, about 4 to24, about 4 to 22, about 4 to 20, about 4 to 18, about 4 to 16, about 4to 14, about 4 to 12, about 4 to 10, about 4 to 8, about 4 to 6, about 6to 36, about 6 to 34, about 6 to 32, about 6 to 30, about 6 to 28, about6 to 26, about 6 to 24, about 6 to 22, about 6 to 20, about 6 to 18,about 6 to 16, about 6 to 14, about 6 to 12, about 6 to 10, about 6 to8, about 8 to 36, about 8 to 34, about 8 to 32, about 8 to 30, about 8to 28, about 8 to 26, about 8 to 24, about 8 to 22, about 8 to 20, about8 to 18, about 8 to 16, about 8 to 14, about 8 to 12, about 8 to 10,about 10 to 36, about 10 to 34, about 10 to 32, about 10 to 30, about 10to 28, about 10 to 26, about 10 to 24, about 10 to 22, about 10 to 20,about 10 to 18, about 10 to 16, about 10 to 14, about 10 to 12, about 12to 36, about 12 to 34, about 12 to 32, about 12 to 30, about 12 to 28,about 12 to 26, about 12 to 24, about 12 to 22, about 12 to 20, about 12to 18, about 12 to 16, about 12 to 14, about 14 to 36, about 14 to 34,about 14 to 32, about 14 to 30, about 14 to 28, about 14 to 26, about 14to 24, about 14 to 22, about 14 to 20, about 14 to 18, about 14 to 16,about 16 to 36, about 16 to 34, about 16 to 32, about 16 to 30, about 16to 28, about 16 to 26, about 16 to 24, about 16 to 22, about 16 to 20,about 16 to 18, about 18 to 36, about 18 to 34, about 18 to 32, about 18to 30, about 18 to 28, about 18 to 26, about 18 to 24, about 18 to 22,about 18 to 20, about 20 to 36, about 20 to 34, about 20 to 32, about 20to 30, about 20 to 28, about 20 to 26, about 20 to 24, about 20 to 22,about 22 to 36, about 22 to 34, about 22 to 32, about 22 to 30, about 22to 28, about 22 to 26, about 22 to 24, about 24 to 36, about 24 to 34,about 24 to 32, about 24 to 30, about 24 to 28, about 24 to 26, about 26to 36, about 26 to 34, about 26 to 32, about 26 to 30, about 26 to 28,about 28 to 36, about 28 to 34, about 28 to 32, about 28 to 30, about 30to 36, about 30 to 34, about 30 to 32, about 32 to 36, about 32 to 34,or about 34 to 36 months.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at refrigeration temperatures (35-40° F.),at room temperature (68-72° F.), between 50-77° F., between −23-35° F.,between 70-100° F., or between 101-213° F. for a period of 1 to 36 1 to34 1 to 32 1 to 30 1 to 28 1 to 26 1 to 24 1 to 22 1 to 20 1 to 18 1 to16 1 to 14 1 to 12 1 to 10 1 to 8 1 to 6 1 one 4 1 to 2 4 to 36 4 to 344 to 32 4 to 30 4 to 28 4 to 26 4 to 24 4 to 22 4 to 20 4 to 18 4 to 164 to 14 4 to 12 4 to 10 4 to 8 4 to 6 6 to 36 6 to 34 6 to 32 6 to 30 6to 28 6 to 26 6 to 24 6 to 22 6 to 20 6 to 18 6 to 16 6 to 14 6 to 12 6to 10 6 to 8 8 to 36 8 to 34 8 to 32 8 to 308 to 28 8 to 26 8 to 24 8 to22 8 to 20 8 to 18 8 to 16 8 to 14 8 to 12 8 to 10 10 to 36 10 to 34 10to 32 10 to 30 10 to 28 10 to 26 10 to 24 10 to 22 10 to 20 10 to 18 10to 16 10 to 14 10 to 12 12 to 36 12 to 34 12 to 32 12 to 30 12 to 28 12to 26 12 to 24 12 to 22 12 to 20 12 to 18 12 to 16 12 to 14 14 to 36 14to 34 14 to 32 14 to 30 14 to 28 14 to 26 14 to 24 14 to 22 14 to 20 14to 18 14 to 16 16 to 36 16 to 34 16 to 32 16 to 30 16 to 28 16 to 26 16to 24 16 to 22 16 to 20 16 to 18 18 to 36 18 to 34 18 to 32 18 to 30 18to 28 18 to 26 18 to 24 18 to 22 18 to 20 20 to 36 20 to 34 20 to 32 20to 30 20 to 28 20 to 26 20 to 24 20 to 22 22 to 36 22 to 34 22 to 32 22to 30 22 to 28 22 to 26 22 to 24 24 to 36 24 to 34 24 to 32 24 to 30 24to 28 24 to 26 26 to 36 26 to 34 26 to 32 26 to 30 26 to 28 28 to 36 28to 34 28 to 32 28 to 30 30 to 36 30 to 34 30 to 32 32 to 36 32 to 34, orabout 34 to 36.

In some embodiments, the microbial compositions of the presentdisclosure are shelf stable at any of the disclosed temperatures and/ortemperature ranges and spans of time at a relative humidity of at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, or 98%

Encapsulation Compositions

In some embodiments, the microbes or microbial compositions of thedisclosure are encapsulated in an encapsulating composition. Anencapsulating composition protects the microbes from external stressorsprior to entering the gastrointestinal tract of ungulates. Encapsulatingcompositions further create an environment that may be beneficial to themicrobes, such as minimizing the oxidative stresses of an aerobicenvironment on anaerobic microbes. See Kalsta et al. (U.S. Pat. No.5,104,662A), Ford (U.S. Pat. No. 5,733,568A), and Mosbach and Nilsson(U.S. Pat. No. 4,647,536A) for encapsulation compositions of microbes,and methods of encapsulating microbes.

in one embodiment, the encapsulating composition comprises microcapsuleshaving a multiplicity of liquid cores encapsulated in a solid shellmaterial. For purposes of the disclosure, a “multiplicity” of cores isdefined as two or more.

A first category of useful fusible shell materials is that of normallysolid fats, including fats which are already of suitable hardness andanimal or vegetable fats and oils which are hydrogenated until theirmelting points are sufficiently high to serve the purposes of thepresent disclosure. Depending on the desired process and storagetemperatures and the specific material selected, a particular fat can beeither a normally solid or normally liquid material. The terms “normallysolid” and “normally liquid” as used herein refer to the state of amaterial at desired temperatures for storing the resultingmicrocapsules. Since fats and hydrogenated oils do not, strictlyspeaking, have melting points, the term “melting point” is used hereinto describe the minimum temperature at which the fusible materialbecomes sufficiently softened or liquid to be successfully emulsifiedand spray cooled, thus roughly corresponding to the maximum temperatureat which the shell material has sufficient integrity to prevent releaseof the choline cores. “Melting point” is similarly defined herein forother materials which do not have a sharp melting point.

Specific examples of fats and oils useful herein (some of which requirehardening) are as follows: animal oils and fats, such as beef tallow,mutton tallow, lamb tallow, lard or pork fat, fish oil, and sperm oil;vegetable oils, such as canola oil, cottonseed oil, peanut oil, cornoil, olive oil, soybean oil, sunflower oil, safflower oil, coconut oil,palm oil, linseed oil, tung oil, and castor oil; fatty acidmonoglycerides and diglycerides; free fatty acids, such as stearic acid,palmitic acid, and oleic acid; and mixtures thereof. The above listingof oils and fats is not meant to be exhaustive, but only exemplary.

Specific examples of fatty acids include linoleic acid, γ-linoleic acid,dihomo-γ-linolenic acid, arachidonic acid, docosatetraenoic acid,vaccenic acid, nervonic acid, mead acid, erucic acid, gondoic acid,elaidic acid, oleic acid, palitoleic acid, stearidonic acid,eicosapentaenoic acid, valeric acid, caproic acid, enanthic acid,caprylic acid, pelargonic acid, capric acid, undecylic acid, lauricacid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid,margaric acid, stearic acid, nonadecyclic acid, arachidic acid,heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid,pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid,nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid,psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid,heptatriacontanoic acid, and octatriacontanoic acid.

Another category of fusible materials useful as encapsulating shellmaterials is that of waxes. Representative waxes contemplated for useherein are as follows: animal waxes, such as beeswax, lanolin, shellwax, and Chinese insect wax; vegetable waxes, such as carnauba,candelilla, bayberry, and sugar cane; mineral waxes, such as paraffin,microcrystalline petroleum, ozocerite, ceresin, and montan; syntheticwaxes, such as low molecular weight polyolefin (e.g., CARBOWAX), andpolyol ether-esters (e.g., sorbitol); Fischer-Tropsch process syntheticwaxes; and mixtures thereof. Water-soluble waxes, such as CARBOWAX andsorbitol, are not contemplated herein if the core is aqueous.

Still other fusible compounds useful herein are fusible natural resins,such as rosin, balsam, shellac, and mixtures thereof.

Various adjunct materials are contemplated for incorporation in fusiblematerials according to the present disclosure. For example,antioxidants, light stabilizers, dyes and lakes, flavors, essentialoils, anti-caking agents, fillers, pH stabilizers, sugars(monosaccharides, disaccharides, trisaccharides, and polysaccharides)and the like can be incorporated in the fusible material in amountswhich do not diminish its utility for the present disclosure.

The core material contemplated herein constitutes from about 0.1% toabout 50%, about 1% to about 35%. or about 5% to about 30% by weight ofthe microcapsules. In some embodiments, the core material contemplatedherein constitutes no more than about 30% by weight of themicrocapsules. In some embodiments, the core material contemplatedherein constitutes about 5% by weight of the microcapsules. The corematerial is contemplated as either a liquid or solid at contemplatedstorage temperatures of the microcapsules.

The cores may include other additives well-known in the pharmaceuticalart, including edible sugars, such as sucrose, glucose, maltose,fructose, lactose, cellobiose, monosaccharides, disaccharides,trisaccharides, polysaccharides, and mixtures thereof; artificialsweeteners, such as aspartame, saccharin, cyclamate salts, and mixturesthereof edible acids, such as acetic acid (vinegar), citric acid,ascorbic acid, tartaric acid, and mixtures thereof; edible starches,such as corn starch; hydrolyzed vegetable protein; water-solublevitamins, such as Vitamin C; water-soluble medicaments; water-solublenutritional materials, such as ferrous sulfate; flavors; salts;monosodium glutamate; antimicrobial agents, such as sorbic acid;antimycotic agents, such as potassium sorbate, sorbic acid, sodiumbenzoate, and benzoic acid; food grade pigments and dyes; and mixturesthereof. Other potentially useful supplemental core materials will beapparent to those of ordinary skill in the art.

Emulsifying agents may be employed to assist in the formation of stableemulsions. Representative emulsifying agents include glycerylmonostearate, polysorbate esters, ethoxylated mono- and diglycerides,and mixtures thereof.

For ease of processing, and particularly to enable the successfulformation of a reasonably stable emulsion, the viscosities of the corematerial and the shell material should be similar at the temperature atwhich the emulsion is formed. In particular, the ratio of the viscosityof the shell to the viscosity of the core, expressed in centipoise orcomparable units, and both measured at the temperature of the emulsion,should be from about 22:1 to about 1:1, desirably from about 8:1 toabout 1:1, and preferably from about 3:1 to about 1:1. A ratio of 1:1would be ideal, but a viscosity ratio within the recited ranges isuseful.

Encapsulating compositions are not limited to microcapsule compositionsas disclosed above. In some embodiments encapsulating compositionsencapsulate the microbial compositions in an adhesive polymer that canbe natural or synthetic without toxic effect. In some embodiments, theencapsulating composition may be a matrix selected from sugar matrix,gelatin matrix, polymer matrix, silica matrix, starch matrix, foammatrix, etc. In some embodiments, the encapsulating composition may beselected from polyvinyl acetates; polyvinyl acetate copolymers; ethylenevinyl acetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcoholcopolymers; celluloses, including ethylcelluloses, methylcelluloses,hydroxymethylcelluloses, hydroxypropylcelluloses andcarboxymethylcellulose; polyvinylpyrolidones; polysaccharides, includingstarch, modified starch, dextrins, maltodextrins, alginate andchitosans; monosaccharides; fats; fatty acids, including oils; proteins,including gelatin and zeins; gum arabics; shellacs; vinylidene chlorideand vinylidene chloride copolymers; calcium lignosulfonates; acryliccopolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymersand copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers;and polychloroprene.

In some embodiments, the encapsulating shell of the present disclosurecan be up to 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540μm, 550 μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630μm, 640 μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720μm, 730 μm, 740 μm, 750 μm, 760 μm, 770 μm, 780 μm, 790 μm, 800 μm, 810μm, 820 μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 880 μm, 890 μm, 900μm, 910 μm, 920 μm, 930 μm, 940 μm, 950 μm, 960 μm, 970 μm, 980 μm, 990μm, 1000 μm, 1010 μm, 1020 μm, 1030 μm, 1040 μm, 1050 μm, 1060 μm, 1070μm, 1080 μm, 1090 μm, 1100 μm, 1110 μm, 1120 μm, 1130 μm, 1140 μm, 1150μm, 1160 μm, 1170 μm, 1180 μm, 1190 μm, 1200 μm, 1210 μm, 1220 μm, 1230μm, 1240 μm, 1250 μm, 1260 μm, 1270 μm, 1280 μm, 1290 μm, 1300 μm, 1310μm, 1320 μm, 1330 μm, 1340 μm, 1350 μm, 1360 μm, 1370 μm, 1380 μm, 1390μm, 1400 μm, 1410 μm, 1420 μm, 1430 μm, 1440 μm, 1450 μm, 1460 μm, 1470μm, 1480 μm, 1490 μm, 1500 μm, 1510 μm, 1520 μm, 1530 μm, 1540 μm, 1550μm, 1560 μm, 1570 μm, 1580 μm, 1590 μm, 1600 μm, 1610 μm, 1620 μm, 1630μm, 1640 μm, 1650 μm, 1660 μm, 1670 μm, 1680 μm, 1690 μm, 1700 μm, 1710μm, 1720 μm, 1730 μm, 1740 μm, 1750 μm, 1760 μm, 1770 μm, 1780 μm, 1790μm, 1800 μm, 1810 μm, 1820 μm, 1830 μm, 1840 μm, 1850 μm, 1860 μm, 1870μm, 1880 μm, 1890 μm, 1900 μm, 1910 μm, 1920 μm, 1930 μm, 1940 μm, 1950μm, 1960 μm, 1970 μm, 1980 μm, 1990 μm, 2000 μm, 2010 μm, 2020 μm, 2030μm, 2040 μm, 2050 μm, 2060 μm, 2070 μm, 2080 μm, 2090 μm, 2100 μm, 2110μm, 2120 μm, 2130 μm, 2140 μm, 2150 μm, 2160 μm, 2170 μm, 2180 μm, 2190μm, 2200 μm, 2210 μm, 2220 μm, 2230 μm, 2240 μm, 2250 μm, 2260 μm, 2270μm, 2280 μm, 2290 μm, 2300 μm, 2310 μm, 2320 μm, 2330 μm, 2340 μm, 2350μm, 2360 μm, 2370 μm, 2380 μm, 2390 μm, 2400 μm, 2410 μm, 2420 μm, 2430μm, 2440 μm, 2450 μm, 2460 μm, 2470 μm, 2480 μm, 2490 μm, 2500 μm, 2510μm, 2520 μm, 2530 μm, 2540 μm, 2550 μm, 2560 μm, 2570 μm, 2580 μm, 2590μm, 2600 μm, 2610 μm, 2620 μm, 2630 μm, 2640 μm, 2650 μm, 2660 μm, 2670μm, 2680 μm, 2690 μm, 2700 μm, 2710 μm, 2720 μm, 2730 μm, 2740 μm, 2750μm, 2760 μm, 2770 μm, 2780 μm, 2790 μm, 2800 μm, 2810 μm, 2820 μm, 2830μm, 2840 μm, 2850 μm, 2860 μm, 2870 μm, 2880 μm, 2890 μm, 2900 μm, 2910μm, 2920 μm, 2930 μm, 2940 μm, 2950 μm, 2960 μm, 2970 μm, 2980 μm, 2990μm, or 3000 μm thick.

Animal Feed

In some embodiments, compositions of the present disclosure are mixedwith animal feed. In some embodiments, animal feed may be present invarious forms such as pellets, capsules, granulated, powdered, liquid,or semi-liquid.

In some embodiments, compositions of the present disclosure are mixedinto the premix at at the feed mill (e.g., Carghill or Western Millin),alone as a standalone premix, and/or alongside other feed additives suchas MONENSIN, vitamins, etc. In one embodiment, the compositions of thepresent disclosure are mixed into the feed at the feed mill. In anotherembodiment, compositions of the present disclosure are mixed into thefeed itself.

In some embodiments, feed of the present disclosure may be supplementedwith water, premix or premixes, forage, fodder, beans (e.g., whole,cracked, or ground), grains (e.g., whole, cracked, or ground), bean- orgrain-based oils, bean- or grain-based meals, bean- or grain-basedhaylage or silage, bean- or grain-based syrups, fatty acids, sugaralcohols (e.g., polyhydric alcohols), commercially available formulafeeds, and mixtures thereof.

In some embodiments, forage encompasses hay, haylage, and silage. Insome embodiments, hays include grass hays (e.g., sudangrass,orchardgrass, or the like), alfalfa hay, and clover hay. In someembodiments, haylages include grass haylages, sorghum haylage, andalfalfa haylage. In some embodiments, silages include maize, oat, wheat,alfalfa, clover, and the like.

In some embodiments, premix or premixes may be utilized in the feed.Premixes may comprise micro-ingredients such as vitamins, minerals,amino acids; chemical preservatives; pharmaceutical compositions such asantibiotics and other medicaments; fermentation products, and otheringredients. In some embodiments, premixes are blended into the feed.

In some embodiments, the feed may include feed concentrates such assoybean hulls, sugar beet pulp, molasses, high protein soybean meal,ground corn, shelled corn, wheat midds, distiller grain, cottonseedhulls, rumen-bypass protein, rumen-bypass fat, and grease. See Luhman(U.S. Publication US20150216817A1), Anderson et al. (U.S. Pat. No.3,484,243) and Porter and Luhman (U.S. Pat. No. 9,179,694B2) for animalfeed and animal feed supplements capable of use in the presentcompositions and methods.

In some embodiments, feed occurs as a compound, which includes, in amixed composition capable of meeting the basic dietary needs, the feeditself, vitamins, minerals, amino acids, and other necessary components.Compound feed may further comprise premixes.

In some embodiments, microbial compositions of the present disclosuremay be mixed with animal feed, premix, and/or compound feed. Individualcomponents of the animal feed may be mixed with the microbialcompositions prior to feeding to ruminants. The microbial compositionsof the present disclosure may be applied into or on a premix, into or ona feed, and/or into or on a compound feed.

Administration of Microbial Compositions

In some embodiments, the microbial compositions of the presentdisclosure are administered to ruminants via the oral route. In someembodiments the microbial compositions are administered via a directinjection route into the gastrointestinal tract. In further embodiments,the direct injection administration delivers the microbial compositionsdirectly to the rumen. In some embodiments, the microbial compositionsof the present disclosure are administered to animals anally. In furtherembodiments, anal administration is in the form of an insertedsuppository.

In some embodiments, the microbial composition is administered in a dosecomprise a total of, or at least, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml,18 ml, 19 ml, 20 ml, 21 ml, 22 ml, 23 ml, 24 ml, 25 ml, 26 ml, 27 ml, 28ml, 29 ml, 30 ml, 31 ml, 32 ml, 33 ml, 34 ml, 35 ml, 36 ml, 37 ml, 38ml, 39 ml, 40 ml, 41 m, 42 ml, 43 ml, 44 ml, 45 ml, 46 ml, 47 ml, 48 ml,49 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, or 1,000 ml.

In some embodiments, the microbial composition is administered in a dosecomprising a total of, or at least, 10¹⁸, 10¹⁷, 10¹⁶, 10¹⁵, 10¹⁴, 10¹³,10¹², 10¹¹, 10¹⁰, 10⁹, 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, or 10² microbialcells.

In some embodiments, the microbial compositions are mixed with feed, andthe administration occurs through the ingestion of the microbialcompositions along with the feed. In some embodiments, the dose of themicrobial composition is administered such that there exists 10² to10¹², 10³ to 10¹², 10⁴ to 10¹², 10⁵ to 10¹², 10⁶ to 10¹², 10⁷ to 10¹²,10⁸ to 10¹², 10⁹ to 10¹², 10¹⁰ to 10¹², 10¹¹ to 10¹², 10² to 10¹¹, 10³to 10¹¹, 10⁴ to 10¹¹, 10⁵ to 10¹¹, 10⁶ to 10¹¹, 10⁷ to 10¹¹, 10⁸ to10¹¹, 10⁹ to 10¹¹, 10¹⁰ to 10¹¹, 10² to 10¹⁰, 10³ to 10¹⁰, 10⁴ to 10¹⁰,10⁵ to 10¹⁰, 10⁶ to 10¹⁰, 10⁷ to 10¹⁰, 10⁸ to 10¹⁰, 10⁹ to 10¹⁰, 10² to10⁹, 10³ to 10⁹, 10⁴ to 10⁹, 10⁵ to 10⁹, 10⁶ to 10⁹, 10⁷ to 10⁹, 10⁸ to10⁹, 10² to 10⁸, 10³ to 10⁸, 10⁴ to 10⁸, 10⁵ to 10⁸, 10⁶ to 10⁸, 10⁷ to10⁸, 10² to 10⁷, 10³ to 10⁷, 10⁴ to 10⁷, 10⁵ to 10⁷, 10⁶ to 10⁷, 10² to10⁶, 10³ to 10⁶, 10⁴ to 10⁶, 10⁵ to 10⁶, 10² to 10⁵, 10³ to 10⁵, 10⁴ to10⁵, 10² to 10⁴, 10³ to 10⁴, 10² to 10³, 10¹², 10¹¹, 10¹⁰, 10⁹, 10⁸,10⁷, 10⁶, 10⁵, 10⁴, 10³, or 10² total microbial cells per gram ormilliliter of the composition.

In some embodiments, the administered dose of the microbial compositioncomprises 10² to 10¹⁸, 10³ to 10¹⁸, 10⁴ to 10¹⁸, 10⁵ to 10¹⁸, 10⁶ to10¹⁸, 10⁷ to 10¹⁸, 10⁸ to 10¹⁸, 10⁹ to 10¹⁸, 10¹⁰ to 10¹⁸, 10¹¹ to 10¹⁸,10¹² to 10¹⁸, 10¹¹ to 10¹⁸, 10¹⁴ to 10¹⁸, 10¹⁵ to 10¹⁸, 10¹⁶ to 10¹⁸,10¹⁷ to 10¹⁸, 10² to 10¹², 10³ to 10¹², 10⁴ to 10¹², 10⁵ to 10¹², 10⁶ to10¹², 10⁷ to 10¹², 10⁸ to 10¹², 10⁹ to 10¹², 10¹⁰ to 10¹², 10¹¹ to 10¹²,10² to 10¹¹, 10³ to 10¹¹, 10⁴ to 10¹¹, 10⁵ to 10¹¹, 10⁶ to 10¹¹, 10⁷ to10¹¹, 10⁸ to 10 ¹¹, 10⁹ to 10¹¹, 10¹⁰ to 10¹¹, 10² to 10¹⁰, 10⁴ to 10¹⁰,10⁵ to 10¹⁰, 10⁶ to 10¹⁰, 10⁷ to 10¹⁰, 10⁸ to 10¹⁰, 10⁹ to 10¹⁰, 10² to10⁹, 10³ to 10⁹, 10⁴ to 10⁹, 10⁵ to 10⁹, 10⁶ to 10⁹, 10⁷ to 10⁹, 10⁸ to10⁹, 10² to 10⁸, 10³ to 10⁸, 10⁴ to 10⁸, 10⁵ to 10⁸, 10⁶ to 10⁸, 10⁷ to10⁸, 10² to 10⁷, 10³ to 10⁷, 10⁴ to 10⁷, 10⁵ to 10⁷, 10⁶ to 10⁷, 10² to10⁶, 10³ to 10⁶, 10⁴ to 10⁶, 10⁵ to 10⁶, 10² to 10⁵, 10³ to 10⁵, 10⁴ to10⁵, 10² to 10⁴, 10³ to 10⁴, 10² to 10³, 10¹⁸, 10¹⁷, 10¹⁶, 10¹⁵, 10¹⁴,10¹³, 10¹², 10¹¹, 10¹⁰, 10⁹, 10⁸, 10⁷, 10⁶, 10⁵, 10⁴, 10³, or 10² totalmicrobial cells.

In some embodiments, the composition is administered 1 or more times perday. In some aspects, the composition is administered with food eachtime the animal is fed. In some embodiments, the composition isadministered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10,4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7,5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day.

In some embodiments, the microbial composition is administered 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10,2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9,3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7,4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times per week.

In some embodiments, the microbial composition is administered 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10,2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9,3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7,4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times per month.

In some embodiments, the microbial composition is administered 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10,2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9,3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7,4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times per year.

In some embodiments, the feed can be uniformly coated with one or morelayers of the microbes and/or microbial compositions disclosed herein,using conventional methods of mixing, spraying, or a combination thereofthrough the use of treatment application equipment that is specificallydesigned and manufactured to accurately, safely, and efficiently applycoatings. Such equipment uses various types of coating technology suchas rotary coaters, drum coaters, fluidized bed techniques, spouted beds,rotary mists, or a combination thereof. Liquid treatments such as thoseof the present disclosure can be applied via either a spinning“atomizer” disk or a spray nozzle, which evenly distributes themicrobial composition onto the feed as it moves though the spraypattern. In some aspects, the feed is then mixed or tumbled for anadditional period of time to achieve additional treatment distributionand drying.

In some embodiments, the feed coats of the present disclosure can be upto 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540 μm, 550μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630 μm, 640μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720 μm, 730μm, 740 μm, 750 μm, 760 μm, 770 μm, 780 μm, 790 μm, 800 μm, 810 μm, 820μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 880 μm, 890 μm, 900 μm, 910μm, 920 μm, 930 μm, 940 μm, 950 μm, 960 μm, 970 μm, 980 μm, 990 μm, 1000μm, 1010 μm, 1020 μm, 1030 μm, 1040 μm, 1050 μm, 1060 μm, 1070 μm, 1080μm, 1090 μm, 1100 μm, 1110 μm, 1120 μm, 1130 μm, 1140 μm, 1150 μm, 1160μm, 1170 μm, 1180 μm, 1190 μm, 1200 μm, 1210 μm, 1220 μm, 1230 μm, 1240μm, 1250 μm, 1260 μm, 1270 μm, 1280 μm, 1290 μm, 1300 μm, 1310 μm, 1320μm, 1330 μm, 1340 μm, 1350 μm, 1360 μm, 1370 μm, 1380 μm, 1390 μm, 1400μm, 1410 μm, 1420 μm, 1430 μm, 1440 μm, 1450 μm, 1460 μm, 1470 μm, 1480μm, 1490 μm, 1500 μm, 1510 μm, 1520 μm, 1530 μm, 1540 μm, 1550 μm, 1560μm, 1570 μm, 1580 μm, 1590 μm, 1600 μm, 1610 μm, 1620 μm, 1630 μm, 1640μm, 1650 μm, 1660 μm, 1670 μm, 1680 μm, 1690 μm, 1700 μm, 1710 μm, 1720μm, 1730 μm, 1740 μm, 1750 μm, 1760 μm, 1770 μm, 1780 μm, 1790 μm, 1800μm, 1810 μm, 1820 μm, 1830 μm, 1840 μm, 1850 μm, 1860 μm, 1870 μm, 1880μm, 1890 μm, 1900 μm, 1910 μm, 1920 μm, 1930 μm, 1940 μm, 1950 μm, 1960μm, 1970 μm, 1980 μm, 1990 μm, 2000 μm, 2010 μm, 2020 μm, 2030 μm, 2040μm, 2050 μm, 2060 μm, 2070 μm, 2080 μm, 2090 μm, 2100 μm, 2110 μm, 2120μm, 2130 μm, 2140 μm, 2150 μm, 2160 μm, 2170 μm, 2180 μm, 2190 μm, 2200μm, 2210 μm, 2220 μm, 2230 μm, 2240 μm, 2250 μm, 2260 μm, 2270 μm, 2280μm, 2290 μm, 2300 μm, 2310 μm, 2320 μm, 2330 μm, 2340 μm, 2350 μm, 2360μm, 2370 μm, 2380 μm, 2390 μm, 2400 μm, 2410 μm, 2420 μm, 2430 μm, 2440μm, 2450 μm, 2460 μm, 2470 μm, 2480 μm, 2490 μm, 2500 μm, 2510 μm, 2520μm, 2530 μm, 2540 μm, 2550 μm, 2560 μm, 2570 μm, 2580 μm, 2590 μm, 2600μm, 2610 μm, 2620 μm, 2630 μm, 2640 μm, 2650 μm, 2660 μm, 2670 μm, 2680μm, 2690 μm, 2700 μm, 2710 μm, 2720 μm, 2730 μm, 2740 μm, 2750 μm, 2760μm, 2770 μm, 2780 μm, 2790 μm, 2800 μm, 2810 μm, 2820 μm, 2830 μm, 2840μm, 2850 μm, 2860 μm, 2870 μm, 2880 μm, 2890 μm, 2900 μm, 2910 μm, 2920μm, 2930 μm, 2940 μm, 2950 μm, 2960 μm, 2970 μm, 2980 μm, 2990 μm, or3000 μm thick.

In some embodiments, the microbial cells can be coated freely onto anynumber of compositions or they can be formulated in a liquid or solidcomposition before being coated onto a composition. For example, a solidcomposition comprising the microorganisms can be prepared by mixing asolid carrier with a suspension of the spores until the solid carriersare impregnated with the spore or cell suspension. This mixture can thenbe dried to obtain the desired particles.

In some other embodiments, it is contemplated that the solid or liquidmicrobial compositions of the present disclosure further containfunctional agents e.g., activated carbon, minerals, vitamins, and otheragents capable of improving the quality of the products or a combinationthereof.

Methods of coating and compositions in use of said methods that areknown in the art can be particularly useful when they are modified bythe addition of one of the embodiments of the present disclosure. Suchcoating methods and apparatus for their application are disclosed in,for example: U.S. Pat. Nos. 8,097,245, and 7,998,502; and PCT Pat. App.Publication Nos. WO 2008/076975, WO 2010/138522, WO 2011/094469, WO2010/111347, and WO 2010/111565 each of which is incorporated byreference herein.

In some embodiments, the microbes or microbial consortia of the presentdisclosure exhibit a synergistic effect, on one or more of the traitsdescribed herein, in the presence of one or more of the microbes orconsortia coming into contact with one another. The synergistic effectobtained by the taught methods can be quantified, for example, accordingto Colby's formula (i.e., (E)=X+Y−(X*Y/100)). See Colby, R. S.,“Calculating Synergistic and Antagonistic Responses of HerbicideCombinations,” 1967. Weeds. Vol. 15, pp. 20-22, incorporated herein byreference in its entirety. Thus, “synergistic” is intended to reflect anoutcome/parameter/effect that has been increased by more than anadditive amount.

In some embodiments, the microbes or microbial consortia of the presentdisclosure may be administered via bolus. In one embodiment, a bolus(e.g., capsule containing the composition) is inserted into a bolus gun,and the bolus gun is inserted into the buccal cavity and/or esophagas ofthe animal, followed by the release/injection of the bolus into theanimal's digestive tract. In one embodiment, the bolus gun/applicator isa BOVIKALC bolus gun/applicator. In another embodiment, the bolusgun/applicator is a QUADRICAL gun/applicator.

In some embodiments, the microbes or microbial consortia of the presentdisclosure may be administered via drench. In one embodiment, the drenchis an oral drench. A drench administration comprises utilizing a drenchkit/applicator/syringe that injects/releases a liquid comprising themicrobes or microbial consortia into the buccal cavity and/or esophagasof the animal.

In some embodiments, the microbes or microbial consortia of the presentdisclosure may be administered in a time-released fashion. Thecomposition may be coated in a chemical composition, or may be containedin a mechanical device or capsule that releases the microbes ormicrobial consortia over a period of time instead all at once. In oneembodiment, the microbes or microbial consortia are administered to ananimal in a time-release capsule. In one embodiment, the composition maybe coated in a chemical composition, or may be contained in a mechanicaldevice or capsul that releases the mcirobes or microbial consortia allat once a period of time hours post ingestion.

In some embodiments, the microbes or microbial consortia areadministered in a time-released fashion between 1 to 5, 1 to 10, 1 to15, 1 to 20, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to90, 1 to 95, or 1 to 100 hours.

In some embodiments, the microbes or microbial consortia areadministered in a time-released fashion between 1 to 2, 1 to 3, 1 to 4,1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to29, or 1 to 30 days.

Microorganisms

As used herein the term “microorganism” should be taken broadly. Itincludes, but is not limited to, the two prokaryotic domains, Bacteriaand Archaea, as well as eukaryotic fungi, protists, and viruses.

By way of example, the microorganisms may include species of the generaof: Clostridium, Ruminococcus, Roseburia, Hydrogenoanaerobacterium,Saccharofermentans, Papillibacter, Pelotomaculum, Butyricicoccus,Tannerella, Prevotella, Butyricimonas, Piromyces, Pichia, Candida,Vrystaatia, Orpinomyces, Neocallimastix, and Phyllosticta. Themicroorganisms may further include species belonging to the family ofLachnospiraceae, and the order of Saccharomycetales. In someembodiments, the microorganisms may include species of any generadisclosed herein.

In certain embodiments, the microorganism is unculturable. This shouldbe taken to mean that the microorganism is not known to be culturable oris difficult to culture using methods known to one skilled in the art.

In one embodiment, the microbes are obtained from animals (e.g.,mammals, reptiles, birds, and the like), soil (e.g., rhizosphere), air,water (e.g., marine, freshwater, wastewater sludge), sediment, oil,plants (e.g., roots, leaves, stems), agricultural products, and extremeenvironments (e.g., acid mine drainage or hydrothermal systems). In afurther embodiment, microbes obtained from marine or freshwaterenvironments such as an ocean, river, or lake. In a further embodiment,the microbes can be from the surface of the body of water, or any depthof the body of water (e.g., a deep sea sample).

The microorganisms of the disclosure may be isolated in substantiallypure or mixed cultures. They may be concentrated, diluted, or providedin the natural concentrations in which they are found in the sourcematerial. For example, microorganisms from saline sediments may beisolated for use in this disclosure by suspending the sediment in freshwater and allowing the sediment to fall to the bottom. The watercontaining the bulk of the microorganisms may be removed by decantationafter a suitable period of settling and either administered to the GItract of an ungulate, or concentrated by filtering or centrifugation,diluted to an appropriate concentration and administered to the GI tractof an ungulate with the bulk of the salt removed. By way of furtherexample, microorganisms from mineralized or toxic sources may besimilarly treated to recover the microbes for application to theungulate to minimize the potential for damage to the animal.

In another embodiment, the microorganisms are used in a crude form, inwhich they are not isolated from the source material in which theynaturally reside. For example, the microorganisms are provided incombination with the source material in which they reside; for example,fecal matter, cud, or other composition found in the gastrointestinaltract. In this embodiment, the source material may include one or morespecies of microorganisms.

In some embodiments, a mixed population of microorganisms is used in themethods of the disclosure.

In embodiments of the disclosure where the microorganisms are isolatedfrom a source material (for example, the material in which theynaturally reside), any one or a combination of a number of standardtechniques which will be readily known to skilled persons may be used.However, by way of example, these in general employ processes by which asolid or liquid culture of a single microorganism can be obtained in asubstantially pure form, usually by physical separation on the surfaceof a solid microbial growth medium or by volumetric dilutive isolationinto a liquid microbial growth medium. These processes may includeisolation from dry material, liquid suspension, slurries or homogenatesin which the material is spread in a thin layer over an appropriatesolid gel growth medium, or serial dilutions of the material made into asterile medium and inoculated into liquid or solid culture media.

Whilst not essential, in one embodiment, the material containing themicroorganisms may be pre-treated prior to the isolation process inorder to either multiply all microorganisms in the material.Microorganisms can then be isolated from the enriched materials asdisclosed above.

In certain embodiments, as mentioned herein before, the microorganism(s)may be used in crude form and need not be isolated from an animal or amedia. For example, cud, feces, or growth media which includes themicroorganisms identified to be of benefit to increased milk productionin ungulates may be obtained and used as a crude source ofmicroorganisms for the next round of the method or as a crude source ofmicroorganisms at the conclusion of the method. For example, fresh fecescould be obtained and optionally processed.

Microbiome Shift and Abundance of Microbes

In some embodiments, the microbiome of a ruminant, including the rumenmicrobiome, comprises a diverse arrive of microbes with a wide varietyof metabolic capabilities. The microbiome is influenced by a range offactors including diet, variations in animal metabolism, and breed,among others. Most bovine diets are plant-based and rich in complexpolysaccharides that enrich the gastrointestinal microbial community formicrobes capable of breaking down specific polymeric components in thediet. The end products of primary degradation sustains a chain ofmicrobes that ultimately produce a range of organic acids together withhydrogen and carbon dioxide. Because of the complex and interlinkednature of the microbiome, changing the diet and thus substrates forprimary degradation may have a cascading effect on rumen microbialmetabolism, with changes in both the organic acid profiles and themethane levels produced, thus impacting the quality and quantity ofanimal production and or the products produced by the animal. SeeMenezes et al. (2011. FEMS Microbiol. Ecol. 78(2):256-265.)

In some aspects, the present disclosure is drawn to administeringmicrobial compositions described herein to modulate or shift themicrobiome of a ruminant.

In some embodiments, the microbiome is shifted through theadministration of one or more microbes to the gastrointestinal tract. Infurther embodiments, the one or more microbes are those selected fromTable 1 or Table 3. In some embodiments, the microbiome shift ormodulation includes a decrease or loss of specific microbes that werepresent prior to the administration of one or more microbes of thepresent disclosure. In some embodiments, the microbiome shift ormodulation includes an increase in microbes that were present prior tothe administration of one or more microbes of the present disclosure. Insome embodiments, the microbiome shift or modulation includes a gain ofone or more microbes that were not present prior to the administrationof one or more microbes of the present disclosure. In a furtherembodiment, the gain of one or more microbes is a microbe that was notspecifically included in the administered microbial consortium.

In some embodiments, the administration of microbes of the presentdisclosure results in a sustained modulation of the microbiome such thatthe administered microbes are present in the microbiome fora period ofat least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10,8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

In some embodiments, the administration of microbes of the presentdisclosure results in a sustained modulation of the microbiome such thatthe administered microbes are present in the microbiome fora period ofat least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10,8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.

In some embodiments, the administration of microbes of the presentdisclosure results in a sustained modulation of the microbiome such thatthe administered microbes are present in the microbiome fora period ofat least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10,8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.

In some embodiments, the presence of the administered microbes aredetected by sampling the gastrointestinal tract and using primers toamplify the 16S or 18S rDNA sequences, or the ITS rDNA sequences of theadministered microbes. In some embodiments, the administered microbesare one or more of those selected from Table 1 or Table 3, and thecorresponding rDNA sequences are those selected from SEQ ID NOs:1-60,SEQ ID NOs:2045-2107 and the SEQ ID NOs identified in Table 3.

In some embodiments, the microbiome of a ruminant is measured byamplifying polynucleotides collected from gastrointestinal samples,wherein the polynucleotides may be 16S or 18S rDNA fragments, or ITSrDNA fragments of microbial rDNA. In one embodiment, the microbiome isfingerprinted by a method of denaturing gradient gel electrophoresis(DGGE) wherein the amplified rDNA fragments are sorted by where theydenature, and form a unique banding pattern in a gel that may be usedfor comparing the microbiome of the same ruminant over time or themicrobiomes of multiple ruminants. In another embodiment, the microbiomeis fingerprinted by a method of terminal restriction fragment lengthpolymorphism (T-RFLP), wherein labelled PCR fragments are digested usinga restriction enzyme and then sorted by size. In a further embodiment,the data collected from the T-RFLP method is evaluated by nonmetricmultidimensional scaling (nMDS) ordination and PERMANOVA statisticsidentify differences in microbiomes, thus allowing for theidentification and measurement of shifts in the microbiome. See alsoShanks et al. (2011. Appl. Environ. Microbiol. 77(9):2992-3001), Petriet al. (2013. PLOS one. 8(12):e83424), and Menezes et al. (2011. FEMSMicrobiol. Ecol. 78(2):256-265.)

In some embodiments, the administration of microbes of the presentdisclosure results in a modulation or shift of the microbiome whichfurther results in a desired phenotype or improved trait.

According to the methods provided herein, a sample is processed todetect the presence of one or more microorganism types in the sample(FIG. 1, 1001; FIG. 2, 2001). The absolute number of one or moremicroorganism organism type in the sample is determined (FIG. 1, 1002;FIG. 2, 2002). The determination of the presence of the one or moreorganism types and the absolute number of at least one organism type canbe conducted in parallel or serially. For example, in the case of asample comprising a microbial community comprising bacteria (i.e., onemicroorganism type) and fungi (i.e., a second microorganism type), theuser in one embodiment detects the presence of one or both of theorganism types in the sample (FIG. 1, 1001; FIG. 2, 2001). The user, ina further embodiment, determines the absolute number of at least oneorganism type in the sample—in the case of this example, the number ofbacteria, fungi or combination thereof, in the sample (FIG. 1, 1002;FIG. 2, 2002).

In one embodiment, the sample, or a portion thereof is subjected to flowcytometry (FC) analysis to detect the presence and/or number of one ormore microorganism types (FIG. 1, 1001, 1002; FIG. 2, 2001, 2002). Inone flow cytometer embodiment, individual microbial cells pass throughan illumination zone, at a rate of at least about 300*s⁻¹, or at leastabout 500*s⁻¹, or at least about 1000*s⁻¹. However, one of ordinaryskill in the art will recognize that this rate can vary depending on thetype of instrument is employed. Detectors which are gated electronicallymeasure the magnitude of a pulse representing the extent of lightscattered. The magnitudes of these pulses are sorted electronically into“bins” or “channels,” permitting the display of histograms of the numberof cells possessing a certain quantitative property (e.g., cell stainingproperty, diameter, cell membrane) versus the channel number. Suchanalysis allows for the determination of the number of cells in each“bin” which in embodiments described herein is an “microorganism type”bin, e.g., a bacteria, fungi, nematode, protozoan, archaea, algae,dinoflagellate, virus, viroid, etc.

In one embodiment, a sample is stained with one or more fluorescent dyeswherein a fluorescent dye is specific to a particular microorganismtype, to enable detection via a flow cytometer or some other detectionand quantification method that harnesses fluorescence, such asfluorescence microscopy. The method can provide quantification of thenumber of cells and/or cell volume of a given organism type in a sample.In a further embodiment, as described herein, flow cytometry isharnessed to determine the presence and quantity of a unique firstmarker and/or unique second marker of the organism type, such as enzymeexpression, cell surface protein expression, etc. Two- or three-variablehistograms or contour plots of, for example, light scattering versusfluorescence from a cell membrane stain (versus fluorescence from aprotein stain or DNA stain) may also be generated, and thus animpression may be gained of the distribution of a variety of propertiesof interest among the cells in the population as a whole. A number ofdisplays of such multiparameter flow cytometric data are in common useand are amenable for use with the methods described herein.

In one embodiment of processing the sample to detect the presence andnumber of one or more microorganism types, a microscopy assay isemployed (FIG. 1, 1001, 1002). In one embodiment, the microscopy isoptical microscopy, where visible light and a system of lenses are usedto magnify images of small samples. Digital images can be captured by acharge-couple device (CCD) camera. Other microscopic techniques include,but are not limited to, scanning electron microscopy and transmissionelectron microscopy. Microorganism types are visualized and quantifiedaccording to the aspects provided herein.

In another embodiment of in order to detect the presence and number ofone or more microorganism types, the sample, or a portion thereof issubjected to fluorescence microscopy. Different fluorescent dyes can beused to directly stain cells in samples and to quantify total cellcounts using an epifluorescence microscope as well as flow cytometry,described above. Useful dyes to quantify microorganisms include but arenot limited to acridine orange (AO), 4,6-di-amino-2 phenylindole (DAPI)and 5-cyano-2,3 Dytolyl Tetrazolium Chloride (CTC). Viable cells can beestimated by a viability staining method such as the LIVE/DEAD°Bacterial Viability Kit (Bac-Light™) which contains two nucleic acidstains: the green-fluorescent SYTO 9™ dye penetrates all membranes andthe red-fluorescent propidium iodide (PI) dye penetrates cells withdamaged membranes. Therefore, cells with compromised membranes willstain red, whereas cells with undamaged membranes will stain green.Fluorescent in situ hybridization (FISH) extends epifluorescencemicroscopy, allowing for the fast detection and enumeration of specificorganisms. FISH uses fluorescent labelled oligonucleotides probes(usually 15-25 basepairs) which bind specifically to organism DNA in thesample, allowing the visualization of the cells using an epifluorescenceor confocal laser scanning microscope (CLSM). Catalyzed reporterdeposition fluorescence in situ hybridization (CARD-FISH) improves uponthe FISH method by using oligonucleotide probes labelled with a horseradish peroxidase (HRP) to amplify the intensity of the signal obtainedfrom the microorganisms being studied. FISH can be combined with othertechniques to characterize microorganism communities. One combinedtechnique is high affinity peptide nucleic acid (PNA)-FISH, where theprobe has an enhanced capability to penetrate through the ExtracellularPolymeric Substance (EPS) matrix. Another example is LIVE/DEAD-FISHwhich combines the cell viability kit with FISH and has been used toassess the efficiency of disinfection in drinking water distributionsystems.

In another embodiment, the sample, or a portion thereof is subjected toRaman micro-spectroscopy in order to determine the presence of amicroorganism type and the absolute number of at least one microorganismtype (FIG. 1, 1001-1002; FIG. 2, 2001-2002). Raman micro-spectroscopy isa non-destructive and label-free technology capable of detecting andmeasuring a single cell Raman spectrum (SCRS). A typical SCRS providesan intrinsic biochemical “fingerprint” of a single cell. A SCRS containsrich information of the biomolecules within it, including nucleic acids,proteins, carbohydrates and lipids, which enables characterization ofdifferent cell species, physiological changes and cell phenotypes. Ramanmicroscopy examines the scattering of laser light by the chemical bondsof different cell biomarkers. A SCRS is a sum of the spectra of all thebiomolecules in one single cell, indicating a cell's phenotypic profile.Cellular phenotypes, as a consequence of gene expression, usuallyreflect genotypes. Thus, under identical growth conditions, differentmicroorganism types give distinct SCRS corresponding to differences intheir genotypes and can thus be identified by their Raman spectra.

In yet another embodiment, the sample, or a portion thereof is subjectedto centrifugation in order to determine the presence of a microorganismtype and the number of at least one microorganism type (FIG. 1,1001-1002; FIG. 2, 2001-2002). This process sediments a heterogeneousmixture by using the centrifugal force created by a centrifuge. Moredense components of the mixture migrate away from the axis of thecentrifuge, while less dense components of the mixture migrate towardsthe axis. Centrifugation can allow fractionation of samples intocytoplasmic, membrane and extracellular portions. It can also be used todetermine localization information for biological molecules of interest.Additionally, centrifugation can be used to fractionate total microbialcommunity DNA. Different prokaryotic groups differ in theirguanine-plus-cytosine (G+C) content of DNA, so density-gradientcentrifugation based on G+C content is a method to differentiateorganism types and the number of cells associated with each type. Thetechnique generates a fractionated profile of the entire community DNAand indicates abundance of DNA as a function of G+C content. The totalcommunity DNA is physically separated into highly purified fractions,each representing a different G+C content that can be analyzed byadditional molecular techniques such as denaturing gradient gelelectrophoresis (DGGE)/amplified ribosomal DNA restriction analysis(ARDRA) (see discussion herein) to assess total microbial communitydiversity and the presence/quantity of one or more microorganism types.

In another embodiment, the sample, or a portion thereof is subjected tostaining in order to determine the presence of a microorganism type andthe number of at least one microorganism type (FIG. 1, 1001-1002; FIG.2, 2001-2002). Stains and dyes can be used to visualize biologicaltissues, cells or organelles within cells. Staining can be used inconjunction with microscopy, flow cytometry or gel electrophoresis tovisualize or mark cells or biological molecules that are unique todifferent microorganism types. In vivo staining is the process of dyeingliving tissues, whereas in vitro staining involves dyeing cells orstructures that have been removed from their biological context.Examples of specific staining techniques for use with the methodsdescribed herein include, but are not limited to: gram staining todetermine gram status of bacteria, endospore staining to identify thepresence of endospores, Ziehl-Neelsen staining, haematoxylin and eosinstaining to examine thin sections of tissue, papanicolaou staining toexamine cell samples from various bodily secretions, periodicacid-Schiff staining of carbohydrates, Masson's trichome employing athree-color staining protocol to distinguish cells from the surroundingconnective tissue, Romanowsky stains (or common variants that includeWright's stain, Jenner's stain, May-Grunwald stain, Leishman stain andGiemsa stain) to examine blood or bone marrow samples, silver stainingto reveal proteins and DNA, Sudan staining for lipids and Conklin'sstaining to detect true endospores. Common biological stains includeacridine orange for cell cycle determination; bismarck brown for acidmucins; carmine for glycogen; carmine alum for nuclei; Coomassie bluefor proteins; Cresyl violet for the acidic components of the neuronalcytoplasm; Crystal violet for cell walls; DAPI for nuclei; eosin forcytoplasmic material, cell membranes, some extracellular structures andred blood cells; ethidium bromide for DNA; acid fuchsine for collagen,smooth muscle or mitochondria; haematoxylin for nuclei; Hoechst stainsfor DNA; iodine for starch; malachite green for bacteria in the Gimenezstaining technique and for spores; methyl green for chromatin; methyleneblue for animal cells; neutral red for Nissl substance; Nile blue fornuclei; Nile red for lipohilic entities; osmium tetroxide for lipids;rhodamine is used in fluorescence microscopy; safranin for nuclei.Stains are also used in transmission electron microscopy to enhancecontrast and include phosphotungstic acid, osmium tetroxide, rutheniumtetroxide, ammonium molybdate, cadmium iodide, carbohydrazide, ferricchloride, hexamine, indium trichloride, lanthanum nitrate, lead acetate,lead citrate, lead(II) nitrate, periodic acid, phosphomolybdic acid,potassium ferricyanide, potassium ferrocyanide, ruthenium red, silvernitrate, silver proteinate, sodium chloroaurate, thallium nitrate,thiosemicarbazide, uranyl acetate, uranyl nitrate, and vanadyl sulfate.

In another embodiment, the sample, or a portion thereof is subjected tomass spectrometry (MS) in order to determine the presence of amicroorganism type and the number of at least one microorganism type(FIG. 1, 1001-1002; FIG. 2, 2001-2002). MS, as discussed below, can alsobe used to detect the presence and expression of one or more uniquemarkers in a sample (FIG. 1, 1003-1004; FIG. 2, 2003-2004). MS is usedfor example, to detect the presence and quantity of protein and/orpeptide markers unique to microorganism types and therefore to providean assessment of the number of the respective microorganism type in thesample. Quantification can be either with stable isotope labelling orlabel-free. De novo sequencing of peptides can also occur directly fromMS/MS spectra or sequence tagging (produce a short tag that can bematched against a database). MS can also reveal post-translationalmodifications of proteins and identify metabolites. MS can be used inconjunction with chromatographic and other separation techniques (suchas gas chromatography, liquid chromatography, capillary electrophoresis,ion mobility) to enhance mass resolution and determination.

In another embodiment, the sample, or a portion thereof is subjected tolipid analysis in order to determine the presence of a microorganismtype and the number of at least one microorganism type (FIG. 1,1001-1002; FIG. 2, 2001-2002). Fatty acids are present in a relativelyconstant proportion of the cell biomass, and signature fatty acids existin microbial cells that can differentiate microorganism types within acommunity. In one embodiment, fatty acids are extracted bysaponification followed by derivatization to give the respective fattyacid methyl esters (FAMEs), which are then analyzed by gaschromatography. The FAME profile in one embodiment is then compared to areference FAME database to identify the fatty acids and theircorresponding microbial signatures by multivariate statistical analyses.

In the aspects of the methods provided herein, the number of uniquefirst makers in the sample, or portion thereof (e.g., sample aliquot) ismeasured, as well as the abundance of each of the unique first markers(FIG. 1, 1003; FIG. 2, 2003). A unique marker is a marker of amicroorganism strain. It should be understood by one of ordinary skillin the art that depending on the unique marker being probed for andmeasured, the entire sample need not be analyzed. For example, if theunique marker is unique to bacterial strains, then the fungal portion ofthe sample need not be analyzed. As described above, in someembodiments, measuring the absolute abundance of one or more organismtypes in a sample comprises separating the sample by organism type,e.g., via flow cytometry.

Any marker that is unique to an organism strain can be employed herein.For example, markers can include, but are not limited to, small subunitribosomal RNA genes (16S/18S rDNA), large subunit ribosomal RNA genes(23S/25S/28S rDNA), intercalary 5.8S gene, cytochrome c oxidase,beta-tubulin, elongation factor, RNA polymerase and internal transcribedspacer (ITS).

Ribosomal RNA genes (rDNA), especially the small subunit ribosomal RNAgenes, i.e., 18S rRNA genes (18S rDNA) in the case of eukaryotes and 16SrRNA (16S rDNA) in the case of prokaryotes, have been the predominanttarget for the assessment of organism types and strains in a microbialcommunity. However, the large subunit ribosomal RNA genes, 28S rDNAs,have been also targeted. rDNAs are suitable for taxonomic identificationbecause: (i) they are ubiquitous in all known organisms; (ii) theypossess both conserved and variable regions; (iii) there is anexponentially expanding database of their sequences available forcomparison. In community analysis of samples, the conserved regionsserve as annealing sites for the corresponding universal PCR and/orsequencing primers, whereas the variable regions can be used forphylogenetic differentiation. In addition, the high copy number of rDNAin the cells facilitates detection from environmental samples.

The internal transcribed spacer (ITS), located between the 18S rDNA and28S rDNA, has also been targeted. The ITS is transcribed but splicedaway before assembly of the ribosomes The ITS region is composed of twohighly variable spacers, ITS1 and ITS2, and the intercalary 5.8S gene.This rDNA operon occurs in multiple copies in genomes. Because the ITSregion does not code for ribosome components, it is highly variable.

In one embodiment, the unique RNA marker can be an mRNA marker, an siRNAmarker or a ribosomal RNA marker.

Protein-coding functional genes can also be used herein as a uniquefirst marker. Such markers include but are not limited to: therecombinase A gene family (bacterial RecA, archaea RadA and RadB,eukaryotic Rad51 and Rad57, phage UvsX); RNA polymerase β subunit (RpoB)gene, which is responsible for transcription initiation and elongation;chaperonins. Candidate marker genes have also been identified forbacteria plus archaea: ribosomal protein S2 (rpsB), ribosomal proteinS10 (rpsJ), ribosomal protein L1 rplA), translation elongation factorEF-2, translation initiation factor IF-2, metalloendopeptidase,ribosomal protein L22, ffh signal recognition particle protein,ribosomal protein L4/Lle (rplD), ribosomal protein L2 (rplB), ribosomalprotein S9 (rpsl), ribosomal protein L3 (rplC), phenylalanyl-tRNAsynthetase beta subunit, ribosomal protein L14b/L23e (rplN), ribosomalprotein S5, ribosomal protein S19 (rpsS), ribosomal protein S7,ribosomal protein L16/L10E (rplP), ribosomal protein S13 (rpsM),phenylalanyl-tRNA synthetase α subunit, ribosomal protein L15, ribosomalprotein L25/L23, ribosomal protein L6 (rplF), ribosomal protein L11(rplK), ribosomal protein L5 (rplE), ribosomal protein S12/S23,ribosomal protein L29, ribosomal protein S3 (rpsC), ribosomal proteinS11 (rpsK), ribosomal protein L10, ribosomal protein S8, tRNApseudouridine synthase B, ribosomal protein L18P/L5E, ribosomal proteinS15P/S13e, Porphobilinogen deaminase, ribosomal protein S17, ribosomalprotein L13 (rplM), phosphoribosylformylglycinamidine cyclo-ligase(rpsE), ribonuclease HII and ribosomal protein L24. Other candidatemarker genes for bacteria include: transcription elongation protein NusA(nusA), rpoB DNA-directed RNA polymerase subunit beta (rpoB),GTP-binding protein EngA, rpoC DNA-directed RNA polymerase subunitbeta′, priA primosome assembly protein, transcription-repair couplingfactor, CTP synthase (pyrG), secY preprotein translocase subunit SecY,GTP-binding protein Obg/CgtA, DNA polymerase I, rpsF 30S ribosomalprotein S6, poA DNA-directed RNA polymerase subunit alpha, peptide chainrelease factor 1, rplI 50S ribosomal protein L9, polyribonucleotidenucleotidyltransferase, tsf elongation factor Ts (tsf), rplQ 50Sribosomal protein L17, tRNA (guanine-N(1)-)-methyltransferase (rplS),rplY probable 50S ribosomal protein L25, DNA repair protein RadA,glucose-inhibited division protein A, ribosome-binding factor A, DNAmismatch repair protein MutL, smpB SsrA-binding protein (smpB),N-acetylglucosaminyl transferase, S-adenosyl-methyltransferase MraW,UDP-N-acetylmuramoylalanine-D-glutamate ligase, rplS 50S ribosomalprotein L19, rp1T 50S ribosomal protein L20 (rplT), ruvA Hollidayjunction DNA helicase, ruvB Holliday junction DNA helicase B, serSseryl-tRNA synthetase, rplU 50S ribosomal protein L21, rpsR 30Sribosomal protein S18, DNA mismatch repair protein MutS, rpsT 30Sribosomal protein S20, DNA repair protein RecN, frr ribosome recyclingfactor (frr), recombination protein RecR, protein of unknown functionUPF0054, miaA tRNA isopentenyltransferase, GTP-binding protein YchF,chromosomal replication initiator protein DnaA, dephospho-CoA kinase,16S rRNA processing protein RimM, ATP-cone domain protein,1-deoxy-D-xylulose 5-phosphate reductoisomerase, 2C-methyl-D-erythritol2,4-cyclodiphosphate synthase, fatty acid/phospholipid synthesis proteinPlsX, tRNA(Ile)-lysidine synthetase, dnaG DNA primase (dnaG), ruvCHolliday junction resolvase, rpsP 30S ribosomal protein S16, RecombinaseA recA, riboflavin biosynthesis protein RibF, glycyl-tRNA synthetasebeta subunit, trmU tRNA(5-methylaminomethyl-2-thiouridylate)-methyltransferase, rpmI 50Sribosomal protein L35, hemE uroporphyrinogen decarboxylase, Rodshape-determining protein, rpmA 50S ribosomal protein L27 (rpmA),peptidyl-tRNA hydrolase, translation initiation factor IF-3 (infC),UDP-N-acetylmuramyl-tripeptide synthetase, rpmF 505 ribosomal proteinL32, rpIL 50S ribosomal protein L7/L12 (rpIL), leuS leucyl-tRNAsynthetase, ligA NAD-dependent DNA ligase, cell division protein FtsA,GTP-binding protein TypA, ATP-dependent Clp protease, ATP-bindingsubunit ClpX, DNA replication and repair protein RecF andUDP-N-acetylenolpyruvoylglucosamine reductase.

Phospholipid fatty acids (PLFAs) may also be used as unique firstmarkers according to the methods described herein. Because PLFAs arerapidly synthesized during microbial growth, are not found in storagemolecules and degrade rapidly during cell death, it provides an accuratecensus of the current living community. All cells contain fatty acids(FAs) that can be extracted and esterified to form fatty acid methylesters (FAMEs). When the FAMEs are analyzed using gaschromatography—mass spectrometry, the resulting profile constitutes a‘fingerprint’ of the microorganisms in the sample. The chemicalcompositions of membranes for organisms in the domains Bacteria andEukarya are comprised of fatty acids linked to the glycerol by anester-type bond (phospholipid fatty acids (PLFAs)). In contrast, themembrane lipids of Archaea are composed of long and branchedhydrocarbons that are joined to glycerol by an ether-type bond(phospholipid ether lipids (PLELs)). This is one of the most widely usednon-genetic criteria to distinguish the three domains. In this context,the phospholipids derived from microbial cell membranes, characterizedby different acyl chains, are excellent signature molecules, becausesuch lipid structural diversity can be linked to specific microbialtaxa.

As provided herein, in order to determine whether an organism strain isactive, the level of expression of one or more unique second markers,which can be the same or different as the first marker, is measured(FIG. 1, 1004; FIG. 2, 2004). Unique first unique markers are describedabove. The unique second marker is a marker of microorganism activity.For example, in one embodiment, the mRNA or protein expression of any ofthe first markers described above is considered a unique second markerfor the purposes of this invention.

In one embodiment, if the level of expression of the second marker isabove a threshold level (e.g., a control level) or at a threshold level,the microorganism is considered to be active (FIG. 1, 1005; FIG. 2,2005). Activity is determined in one embodiment, if the level ofexpression of the second marker is altered by at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, or at least about 30%, as compared to a threshold level, which insome embodiments, is a control level.

Second unique markers are measured, in one embodiment, at the protein,RNA or metabolite level. A unique second marker is the same or differentas the first unique marker.

As provided above, a number of unique first markers and unique secondmarkers can be detected according to the methods described herein.Moreover, the detection and quantification of a unique first marker iscarried out according to methods known to those of ordinary skill in theart (FIG. 1, 1003-1004, FIG. 2, 2003-2004).

Nucleic acid sequencing (e.g., gDNA, cDNA, rRNA, mRNA) in one embodimentis used to determine absolute abundance of a unique first marker and/orunique second marker. Sequencing platforms include, but are not limitedto, Sanger sequencing and high-throughput sequencing methods availablefrom Roche/454 Life Sciences, Illumina/Solexa, Pacific Biosciences, IonTorrent and Nanopore. The sequencing can be amplicon sequencing ofparticular DNA or RNA sequences or whole metagenome/transcriptomeshotgun sequencing.

Traditional Sanger sequencing (Sanger et al. (1977) DNA sequencing withchain-terminating inhibitors. Proc Natl. Acad. Sci. USA, 74, pp.5463-5467, incorporated by reference herein in its entirety) relies onthe selective incorporation of chain-terminating dideoxynucleotides byDNA polymerase during in vitro DNA replication and is amenable for usewith the methods described herein.

In another embodiment, the sample, or a portion thereof is subjected toextraction of nucleic acids, amplification of DNA of interest (such asthe rRNA gene) with suitable primers and the construction of clonelibraries using sequencing vectors. Selected clones are then sequencedby Sanger sequencing and the nucleotide sequence of the DNA of interestis retrieved, allowing calculation of the number of unique microorganismstrains in a sample.

454 pyrosequencing from Roche/454 Life Sciences yields long reads andcan be harnessed in the methods described herein (Margulies et al.(2005) Nature, 437, pp. 376-380; U.S. Pat. Nos. 6,274,320; 6,258,568;6,210,891, each of which is herein incorporated in its entirety for allpurposes). Nucleic acid to be sequenced (e.g., amplicons or nebulizedgenomic/metagenomic DNA) have specific adapters affixed on either end byPCR or by ligation. The DNA with adapters is fixed to tiny beads(ideally, one bead will have one DNA fragment) that are suspended in awater-in-oil emulsion. An emulsion PCR step is then performed to makemultiple copies of each DNA fragment, resulting in a set of beads inwhich each bead contains many cloned copies of the same DNA fragment.Each bead is then placed into a well of a fiber-optic chip that alsocontains enzymes necessary for the sequencing-by-synthesis reactions.The addition of bases (such as A, C, G, or T) trigger pyrophosphaterelease, which produces flashes of light that are recorded to infer thesequence of the DNA fragments in each well. About 1 million reads perrun with reads up to 1,000 bases in length can be achieved. Paired-endsequencing can be done, which produces pairs of reads, each of whichbegins at one end of a given DNA fragment. A molecular barcode can becreated and placed between the adapter sequence and the sequence ofinterest in multiplex reactions, allowing each sequence to be assignedto a sample bioinformatically.

Illumina/Solexa sequencing produces average read lengths of about 25basepairs (bp) to about 300 bp (Bennett et al. (2005) Pharmacogenomics,6:373-382; Lange et al. (2014). BMC Genomics 15, p. 63; Fadrosh et al.(2014) Microbiome 2, p. 6; Caporaso et al. (2012) ISME J, 6, p.1621-1624; Bentley et al. (2008) Accurate whole human genome sequencingusing reversible terminator chemistry. Nature, 456:53-59). Thissequencing technology is also sequencing-by-synthesis but employsreversible dye terminators and a flow cell with a field of oligosattached. DNA fragments to be sequenced have specific adapters on eitherend and are washed over a flow cell filled with specificoligonucleotides that hybridize to the ends of the fragments. Eachfragment is then replicated to make a cluster of identical fragments.Reversible dye-terminator nucleotides are then washed over the flow celland given time to attach. The excess nucleotides are washed away, theflow cell is imaged, and the reversible terminators can be removed sothat the process can repeat and nucleotides can continue to be added insubsequent cycles. Paired-end reads that are 300 bases in length eachcan be achieved. An Illumina platform can produce 4 billion fragments ina paired-end fashion with 125 bases for each read in a single run.Barcodes can also be used for sample multiplexing, but indexing primersare used.

The SOLiD (Sequencing by Oligonucleotide Ligation and Detection, LifeTechnologies) process is a “sequencing-by-ligation” approach, and can beused with the methods described herein for detecting the presence andabundance of a first marker and/or a second marker (FIG. 1, 1003-1004;FIG. 2, 2003-2004) (Peckham et al. SOLiD™ Sequencing and 2-BaseEncoding. San Diego, Calif.: American Society of Human Genetics, 2007;Mitra et al. (2013) Analysis of the intestinal microbiota using SOLiD16S rRNA gene sequencing and SOLiD shotgun sequencing. BMC Genomics,14(Suppl 5): S16; Mardis (2008) Next-generation DNA sequencing methods.Annu Rev Genomics Hum Genet, 9:387-402; each incorporated by referenceherein in its entirety). A library of DNA fragments is prepared from thesample to be sequenced, and are used to prepare clonal bead populations,where only one species of fragment will be present on the surface ofeach magnetic bead. The fragments attached to the magnetic beads willhave a universal P1 adapter sequence so that the starting sequence ofevery fragment is both known and identical. Primers hybridize to the P1adapter sequence within the library template. A set of fourfluorescently labelled di-base probes compete for ligation to thesequencing primer. Specificity of the di-base probe is achieved byinterrogating every 1st and 2nd base in each ligation reaction. Multiplecycles of ligation, detection and cleavage are performed with the numberof cycles determining the eventual read length. The SOLiD platform canproduce up to 3 billion reads per run with reads that are 75 bases long.Paired-end sequencing is available and can be used herein, but with thesecond read in the pair being only 35 bases long. Multiplexing ofsamples is possible through a system akin to the one used by Illumina,with a separate indexing run.

The Ion Torrent system, like 454 sequencing, is amenable for use withthe methods described herein for detecting the presence and abundance ofa first marker and/or a second marker (FIG. 1, 1003-1004; FIG. 2,2003-2004). It uses a plate of microwells containing beads to which DNAfragments are attached. It differs from all of the other systems,however, in the manner in which base incorporation is detected. When abase is added to a growing DNA strand, a proton is released, whichslightly alters the surrounding pH. Microdetectors sensitive to pH areassociated with the wells on the plate, and they record when thesechanges occur. The different bases (A, C, G, T) are washed sequentiallythrough the wells, allowing the sequence from each well to be inferred.The Ion Proton platform can produce up to 50 million reads per run thathave read lengths of 200 bases. The Personal Genome Machine platform haslonger reads at 400 bases. Bidirectional sequencing is available.Multiplexing is possible through the standard in-line molecular barcodesequencing.

Pacific Biosciences (PacBio) SMRT sequencing uses a single-molecule,real-time sequencing approach and in one embodiment, is used with themethods described herein for detecting the presence and abundance of afirst marker and/or a second marker (FIG. 1, 1003-1004; FIG. 2,2003-2004). The PacBio sequencing system involves no amplification step,setting it apart from the other major next-generation sequencingsystems. In one embodiment, the sequencing is performed on a chipcontaining many zero-mode waveguide (ZMW) detectors. DNA polymerases areattached to the ZMW detectors and phospholinked dye-labeled nucleotideincorporation is imaged in real time as DNA strands are synthesized. ThePacBio system yields very long read lengths (averaging around 4,600bases) and a very high number of reads per run (about 47,000). Thetypical “paired-end” approach is not used with PacBio, since reads aretypically long enough that fragments, through CCS, can be coveredmultiple times without having to sequence from each end independently.Multiplexing with PacBio does not involve an independent read, butrather follows the standard “in-line” barcoding model.

In one embodiment, where the first unique marker is the ITS genomicregion, automated ribosomal intergenic spacer analysis (ARISA) is usedin one embodiment to determine the number and identity of microorganismstrains in a sample (FIG. 1, 1003, FIG. 2, 2003) (Ranjard et al. (2003).Environmental Microbiology 5, pp. 1111-1120, incorporated by referencein its entirety for all puposes). The ITS region has significantheterogeneity in both length and nucleotide sequence. The use of afluorescence-labeled forward primer and an automatic DNA sequencerpermits high resolution of separation and high throughput. The inclusionof an internal standard in each sample provides accuracy in sizinggeneral fragments.

In another embodiment, fragment length polymorphism (RFLP) ofPCR-amplified rDNA fragments, otherwise known as amplified ribosomal DNArestriction analysis (ARDRA), is used to characterize unique firstmarkers and the abundance of the same in samples (FIG. 1, 1003, FIG. 2,2003) (Massol-Deya et al. (1995). Mol. Microb. Ecol. Manual. 3.3.2, pp.1-18, incorporated by reference in its entirety for all puposes). rDNAfragments are generated by PCR using general primers, digested withrestriction enzymes, electrophoresed in agarose or acrylamide gels, andstained with ethidium bromide or silver nitrate.

One fingerprinting technique used in detecting the presence andabundance of a unique first marker is single-stranded-conformationpolymorphism (SSCP) (Lee et al. (1996). Appl Environ Microbiol 62, pp.3112-3120; Scheinert et al. (1996). J. Microbiol. Methods 26, pp.103-117; Schwieger and Tebbe (1998). Appl. Environ. Microbiol. 64, pp.4870-4876, each of which is incorporated by reference herein in itsentirety). In this technique, DNA fragments such as PCR productsobtained with primers specific for the 16S rRNA gene, are denatured anddirectly electrophoresed on a non-denaturing gel. Separation is based ondifferences in size and in the folded conformation of single-strandedDNA, which influences the electrophoretic mobility. Reannealing of DNAstrands during electrophoresis can be prevented by a number ofstrategies, including the use of one phosphorylated primer in the PCRfollowed by specific digestion of the phosphorylated strands with lambdaexonuclease and the use of one biotinylated primer to perform magneticseparation of one single strand after denaturation. To assess theidentity of the predominant populations in a given consortium, in oneembodiment, bands are excised and sequenced, or SSCP-patterns can behybridized with specific probes. Electrophoretic conditions, such as gelmatrix, temperature, and addition of glycerol to the gel, can influencethe separation.

In addition to sequencing based methods, other methods for quantifyingexpression (e.g., gene, protein expression) of a second marker areamenable for use with the methods provided herein for determining thelevel of expression of one or more second markers (FIG. 1, 1004; FIG. 2,2004). For example, quantitative RT-PCR, microarray analysis, linearamplification techniques such as nucleic acid sequence basedamplification (NASBA) are all amenable for use with the methodsdescribed herein, and can be carried out according to methods known tothose of ordinary skill in the art.

In another embodiment, the sample, or a portion thereof is subjected toa quantitative polymerase chain reaction (PCR) for detecting thepresence and abundance of a first marker and/or a second marker (FIG. 1,1003-1004; FIG. 2, 2003-2004). Specific microorganism strains activityis measured by reverse transcription of transcribed ribosomal and/ormessenger RNA (rRNA and mRNA) into complementary DNA (cDNA), followed byPCR (RT-PCR).

In another embodiment, the sample, or a portion thereof is subjected toPCR-based fingerprinting techniques to detect the presence and abundanceof a first marker and/or a second marker (FIG. 1, 1003-1004; FIG. 2,2003-2004). PCR products can be separated by electrophoresis based onthe nucleotide composition. Sequence variation among the different DNAmolecules influences the melting behaviour, and therefore molecules withdifferent sequences will stop migrating at different positions in thegel. Thus electrophoretic profiles can be defined by the position andthe relative intensity of different bands or peaks and can be translatedto numerical data for calculation of diversity indices. Bands can alsobe excised from the gel and subsequently sequenced to reveal thephylogenetic affiliation of the community members. Electrophoresismethods include, but are not limited to: denaturing gradient gelelectrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE),single-stranded-conformation polymorphism (SSCP), restriction fragmentlength polymorphism analysis (RFLP) or amplified ribosomal DNArestriction analysis (ARDRA), terminal restriction fragment lengthpolymorphism analysis (T-RFLP), automated ribosomal intergenic spaceranalysis (ARISA), randomly amplified polymorphic DNA (RAPD), DNAamplification fingerprinting (DAF) and Bb-PEG electrophoresis.

In another embodiment, the sample, or a portion thereof is subjected toa chip-based platform such as microarray or microfluidics to determinethe abundance of a unique first marker and/or presence/abundance of aunique second marker (FIG. 1, 1003-1004, FIG. 2, 2003-2004). The PCRproducts are amplified from total DNA in the sample and directlyhybridized to known molecular probes affixed to microarrays. After thefluorescently labeled PCR amplicons are hybridized to the probes,positive signals are scored by the use of confocal laser scanningmicroscopy. The microarray technique allows samples to be rapidlyevaluated with replication, which is a significant advantage inmicrobial community analyses. In general, the hybridization signalintensity on microarrays is directly proportional to the abundance ofthe target organism. The universal high-density 16S microarray(PhyloChip) contains about 30,000 probes of 16SrRNA gene targeted toseveral cultured microbial species and “candidate divisions”. Theseprobes target all 121 demarcated prokaryotic orders and allowsimultaneous detection of 8,741 bacterial and archaeal taxa. Anothermicroarray in use for profiling microbial communities is the

Functional Gene Array (FGA). Unlike PhyloChips, FGAs are designedprimarily to detect specific metabolic groups of bacteria. Thus, FGA notonly reveal the community structure, but they also shed light on the insitu community metabolic potential. FGA contain probes from genes withknown biological functions, so they are useful in linking microbialcommunity composition to ecosystem functions. An FGA termed GeoChipcontains >24,000 probes from all known metabolic genes involved invarious biogeochemical, ecological, and environmental processes such asammonia oxidation, methane oxidation, and nitrogen fixation.

A protein expression assay, in one embodiment, is used with the methodsdescribed herein for determining the level of expression of one or moresecond markers (FIG. 1, 1004; FIG. 2, 2004). For example, in oneembodiment, mass spectrometry or an immunoassay such as an enzyme-linkedimmunosorbant assay (ELISA) is utilized to quantify the level ofexpression of one or more unique second markers, wherein the one or moreunique second markers is a protein.

In one embodiment, the sample, or a portion thereof is subjected toBromodeoxyuridine (BrdU) incorporation to determine the level of asecond unique marker (FIG. 1, 1004; FIG. 2, 2004). BrdU, a syntheticnucleoside analog of thymidine, can be incorporated into newlysynthesized DNA of replicating cells. Antibodies specific for BRdU canthen be used for detection of the base analog. Thus BrdU incorporationidentifies cells that are actively replicating their DNA, a measure ofactivity of a microorganism according to one embodiment of the methodsdescribed herein. BrdU incorporation can be used in combination withFISH to provide the identity and activity of targeted cells.

In one embodiment, the sample, or a portion thereof is subjected tomicroautoradiography (MAR) combined with FISH to determine the level ofa second unique marker (FIG. 1, 1004; FIG. 2, 2004). MAR-FISH is basedon the incorporation of radioactive substrate into cells, detection ofthe active cells using autoradiography and identification of the cellsusing FISH. The detection and identification of active cells atsingle-cell resolution is performed with a microscope. MAR-FISH providesinformation on total cells, probe targeted cells and the percentage ofcells that incorporate a given radiolabelled substance. The methodprovides an assessment of the in situ function of targetedmicroorganisms and is an effective approach to study the in vivophysiology of microorganisms. A technique developed for quantificationof cell-specific substrate uptake in combination with MAR-FISH is knownas quantitative MAR (QMAR).

In one embodiment, the sample, or a portion thereof is subjected tostable isotope Raman spectroscopy combined with FISH (Raman-FISH) todetermine the level of a second unique marker (FIG. 1, 1004; FIG. 2,2004). This technique combines stable isotope probing, Ramanspectroscopy and FISH to link metabolic processes with particularorganisms. The proportion of stable isotope incorporation by cellsaffects the light scatter, resulting in measurable peak shifts forlabelled cellular components, including protein and mRNA components.Raman spectroscopy can be used to identify whether a cell synthesizescompounds including, but not limited to: oil (such as alkanes), lipids(such as triacylglycerols (TAG)), specific proteins (such as hemeproteins, metalloproteins), cytochrome (such as P450, cytochrome c),chlorophyll, chromophores (such as pigments for light harvestingcarotenoids and rhodopsins), organic polymers (such aspolyhydroxyalkanoates (PHA), polyhydroxybutyrate (PHB)), hopanoids,steroids, starch, sulfide, sulfate and secondary metabolites (such asvitamin B12).

In one embodiment, the sample, or a portion thereof is subjected toDNA/RNA stable isotope probing (SIP) to determine the level of a secondunique marker (FIG. 1, 1004; FIG. 2, 2004). SIP enables determination ofthe microbial diversity associated with specific metabolic pathways andhas been generally applied to study microorganisms involved in theutilization of carbon and nitrogen compounds. The substrate of interestis labelled with stable isotopes (such as ¹³C or ¹⁵N) and added to thesample. Only microorganisms able to metabolize the substrate willincorporate it into their cells. Subsequently, ¹³C-DNA and ¹⁵N-DNA canbe isolated by density gradient centrifugation and used for metagenomicanalysis. RNA-based SIP can be a responsive biomarker for use in SIPstudies, since RNA itself is a reflection of cellular activity.

In one embodiment, the sample, or a portion thereof is subjected toisotope array to determine the level of a second unique marker (FIG. 1,1004; FIG. 2, 2004). Isotope arrays allow for functional andphylogenetic screening of active microbial communities in ahigh-throughput fashion. The technique uses a combination of SIP formonitoring the substrate uptake profiles and microarray technology fordetermining the taxonomic identities of active microbial communities.Samples are incubated with a ¹⁴C-labeled substrate, which during thecourse of growth becomes incorporated into microbial biomass. The¹⁴C-labeled rRNA is separated from unlabeled rRNA and then labeled withfluorochromes. Fluorescent labeled rRNA is hybridized to a phylogeneticmicroarray followed by scanning for radioactive and fluorescent signals.The technique thus allows simultaneous study of microbial communitycomposition and specific substrate consumption by metabolically activemicroorganisms of complex microbial communities.

In one embodiment, the sample, or a portion thereof is subjected to ametabolomics assay to determine the level of a second unique marker(FIG. 1, 1004; FIG. 2, 2004). Metabolomics studies the metabolome whichrepresents the collection of all metabolites, the end products ofcellular processes, in a biological cell, tissue, organ or organism.This methodology can be used to monitor the presence of microorganismsand/or microbial mediated processes since it allows associating specificmetabolite profiles with different microorganisms. Profiles ofintracellular and extracellular metabolites associated with microbialactivity can be obtained using techniques such as gaschromatography-mass spectrometry (GC-MS). The complex mixture of ametabolomic sample can be separated by such techniques as gaschromatography, high performance liquid chromatography and capillaryelectrophoresis. Detection of metabolites can be by mass spectrometry,nuclear magnetic resonance (NMR) spectroscopy, ion-mobilityspectrometry, electrochemical detection (coupled to HPLC) and radiolabel(when combined with thin-layer chromatography).

According to the embodiments described herein, the presence andrespective number of one or more active microorganism strains in asample are determined (FIG. 1, 1006; FIG. 2, 2006). For example, strainidentity information obtained from assaying the number and presence offirst markers is analyzed to determine how many occurrences of a uniquefirst marker are present, thereby representing a unique microorganismstrain (e.g., by counting the number of sequence reads in a sequencingassay). This value can be represented in one embodiment as a percentageof total sequence reads of the first maker to give a percentage ofunique microorganism strains of a particular microorganism type. In afurther embodiment, this percentage is multiplied by the number ofmicroorganism types (obtained at step 1002 or 2002, see FIG. 1 and FIG.2) to give the absolute abundance of the one or more microorganismstrains in a sample and a given volume.

The one or more microorganism strains are considered active, asdescribed above, if the level of second unique marker expression at athreshold level, higher than a threshold value, e.g., higher than atleast about 5%, at least about 10%, at least about 20% or at least about30% over a control level.

In another aspect of the invention, a method for determining theabsolute abundance of one or more microorganism strains is determined ina plurality of samples (FIG. 2, see in particular, 2007). For amicroorganism strain to be classified as active, it need only be activein one of the samples. The samples can be taken over multiple timepoints from the same source, or can be from different environmentalsources (e.g., different animals).

The absolute abundance values over samples are used in one embodiment torelate the one or more active microorganism strains, with anenvironmental parameter (FIG. 2, 2008). In one embodiment, theenvironmental parameter is the presence of a second active microorganismstrain. Relating the one or more active microorganism strains to theenvironmental parameter, in one embodiment, is carried out bydetermining the co-occurrence of the strain and parameter by correlationor by network analysis.

In one embodiment, determining the co-occurrence of one or more activemicroorganism strains with an environmental parameter comprises anetwork and/or cluster analysis method to measure connectivity ofstrains or a strain with an environmental parameter within a network,wherein the network is a collection of two or more samples that share acommon or similar environmental parameter. In another embodiment, thenetwork and/or cluster analysis method may be applied to determining theco-occurrence of two or more active microorganism strains in a sample(FIG. 2, 2008). In another embodiment, the network analysis comprisesnonparametric approaches including mutual information to establishconnectivity between variables. In another embodiment, the networkanalysis comprises linkage analysis, modularity analysis, robustnessmeasures, betweenness measures, connectivity measures, transitivitymeasures, centrality measures or a combination thereof (FIG. 2, 2009).In another embodiment, the cluster analysis method comprises building aconnectivity model, subspace model, distribution model, density model,or a centroid model and/or using community detection algorithms such asthe Louvain, Bron-Kerbosch, Girvan-Newman, Clauset-Newman-Moore,Pons-Latapy, and Wakita-Tsurumi algorithms (FIG. 2, 2010).

In one embodiment, the cluster analysis method is a heuristic methodbased on modularity optimization. In a further embodiment, the clusteranalysis method is the Louvain method. See, e.g., the method describedby Blondel et al. (2008). Fast unfolding of communities in largenetworks. Journal of Statistical Mechanics: Theory and Experiment,Volume 2008, October 2008, incorporated by reference herein in itsentirety for all purposes.

In another embodiment, the network analysis comprises predictivemodeling of network through link mining and prediction, collectiveclassification, link-based clustering, relational similarity, or acombination thereof. In another embodiment, the network analysiscomprises differential equation based modeling of populations. Inanother embodiment, the network analysis comprises Lotka-Volterramodeling.

In one embodiment, relating the one or more active microorganism strainsto an environmental parameter (e.g., determining the co-occurrence) inthe sample comprises creating matrices populated with linkages denotingenvironmental parameter and microorganism strain associations.

In one embodiment, the multiple sample data obtained at step 2007 (e.g.,over two or more samples which can be collected at two or more timepoints where each time point corresponds to an individual sample), iscompiled. In a further embodiment, the number of cells of each of theone or more microorganism strains in each sample is stored in anassociation matrix (which can be in some embodiments, an abundancematrix). In one embodiment, the association matrix is used to identifyassociations between active microorganism strains in a specific timepoint sample using rule mining approaches weighted with association(e.g., abundance) data. Filters are applied in one embodiment to removeinsignificant rules.

In one embodiment, the absolute abundance of one or more, or two or moreactive microorganism strains is related to one or more environmentalparameters (FIG. 2, 2008), e.g., via co-occurrence determination.Environmental parameters are chosen by the user depending on thesample(s) to be analyzed and are not restricted by the methods describedherein. The environmental parameter can be a parameter of the sampleitself, e.g., pH, temperature, amount of protein in the sample.Alternatively, the environmental parameter is a parameter that affects achange in the identity of a microbial community (i.e., where the“identity” of a microbial community is characterized by the type ofmicroorganism strains and/or number of particular microorganism strainsin a community), or is affected by a change in the identity of amicrobial community. For example, an environmental parameter in oneembodiment, is the food intake of an animal or the amount of milk (orthe protein or fat content of the milk) produced by a lactating ruminantIn one embodiment, the environmental parameter is the presence, activityand/or abundance of a second microorganism strain in the microbialcommunity, present in the same sample.

In some embodiments described herein, an environmental parameter isreferred to as a metadata parameter.

Other examples of metadata parameters include but are not limited togenetic information from the host from which the sample was obtained(e.g., DNA mutation information), sample pH, sample temperature,expression of a particular protein or mRNA, nutrient conditions (e.g.,level and/or identity of one or more nutrients) of the surroundingenvironment/ecosystem), susceptibility or resistance to disease, onsetor progression of disease, susceptibility or resistance of the sample totoxins, efficacy of xenobiotic compounds (pharmaceutical drugs),biosynthesis of natural products, or a combination thereof.

For example, according to one embodiment, microorganism strain numberchanges are calculated over multiple samples according to the method ofFIG. 2 (i.e., at 2001-2007). Strain number changes of one or more activestrains over time is compiled (e.g., one or more strains that haveinitially been identified as active according to step 2006), and thedirectionality of change is noted (i.e., negative values denotingdecreases, positive values denoting increases). The number of cells overtime is represented as a network, with microorganism strainsrepresenting nodes and the abundance weighted rules representing edges.Markov chains and random walks are leveraged to determine connectivitybetween nodes and to define clusters. Clusters in one embodiment arefiltered using metadata in order to identify clusters associated withdesirable metadata (FIG. 2, 2008).

In a further embodiment, microorganism strains are ranked according toimportance by integrating cell number changes over time and strainspresent in target clusters, with the highest changes in cell numberranking the highest.

Network and/or cluster analysis method in one embodiment, is used tomeasure connectivity of the one or more strains within a network,wherein the network is a collection of two or more samples that share acommon or similar environmental parameter. In one embodiment, networkanalysis comprises linkage analysis, modularity analysis, robustnessmeasures, betweenness measures, connectivity measures, transitivitymeasures, centrality measures or a combination thereof. In anotherembodiment, network analysis comprises predictive modeling of networkthrough link mining and prediction, social network theory, collectiveclassification, link-based clustering, relational similarity, or acombination thereof. In another embodiment, network analysis comprisesdifferential equation based modeling of populations. In yet anotherembodiment, network analysis comprises Lotka-Volterra modeling.

Cluster analysis method comprises building a connectivity model,subspace model, distribution model, density model, or a centroid model.

Network and cluster based analysis, for example, to carry out methodstep 2008 of FIG. 2, can be carried out via a module. As used herein, amodule can be, for example, any assembly, instructions and/or set ofoperatively-coupled electrical components, and can include, for example,a memory, a processor, electrical traces, optical connectors, software(executing in hardware) and/or the like.

Bovine Pathogen Resistance and Clearance

In some aspects, the present disclosure is drawn to administering one ormore microbial compositions described herein to cows to clear thegastrointestinal tract of pathogenic microbes. In some embodiments, thepresent disclosure is further drawn to administering microbialcompositions described herein to prevent colonization of pathogenicmicrobes in the gastrointestinal tract. In some embodiments, theadministration of microbial compositions described herein further clearspathogens from the integument and the respiratory tract of cows, and/orprevent colonization of pathogens on the integument and in therespiratory tract. In some embodiments, the administration of microbialcompositions described herein reduce leaky gut/intestinal permeability,inflammation, and/or incidence of liver disease.

In some embodiments, the microbial compositions of the presentdisclosure comprise one or more microbes that are present in thegastrointestinal tract of cows at a relative abundance of less than 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%,or 0.01%.

In some embodiments, after administration of microbial compositions ofthe present disclosure the one or more microbes are present in thegastrointestinal tract of the cow at a relative abundance of at least0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95%.

Pathogenic microbes of cows may include the following: Clostridiumperfringens, Clostridium botulinum, Salmonella typi, Salmonellatyphimurium, Salmonella enterica, Salmonella pullorum, Erysipelothrixinsidiosa, Campylobacter jejuni, Campylobacter coli, Campylobacter lari,Listeria monocytogenes, Streptococcus agalactiae, Streptococcusdysgalactiae, Corynebacterium bovis, Mycoplasma sp., Citrobacter sp.,Enterobacter sp., Pseudomonas aeruginosa, Pasteurella sp., Bacilluscereus, Bacillus licheniformis, Streptococcus uberis, Staphylococcusaureus, and pathogenic strains of Escherichia coli and Staphylococcusaureus. In some embodiments, the pathogenic microbes include viralpathogens. In some embodiments, the pathogenic microbes are pathogenicto both cows and humans. In some embodiments, the pathogenic microbesare pathogenic to either cows or humans.

In some embodiments, the administration of compositions of the presentdisclosure to cows modulate the makeup of the gastrointestinalmicrobiome such that the administered microbes outcompete microbialpathogens present in the gastrointestinal tract. In some embodiments,the administration of compositions of the present disclosure to cowsharboring microbial pathogens outcompetes the pathogens and clears cowsof the pathogens. In some embodiments, the administration ofcompositions of the present disclosure results in the stimulation ofhost immunity, and aid in clearance of the microbial pathogens. In someembodiments, the administration of compositions of the presentdisclosure introduce microbes that produce bacteriostatic and/orbactericidal components that decrease or clear the cows of the microbialpathogens. (U.S. Pat. No. 8,345,010).

In some embodiments, challenging cows with a microbial colonizer ormicrobial pathogen after administering one or more compositions of thepresent disclosure prevents the microbial colonizer or microbialpathogen from growing to a relative abundance of greater than 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or0.01%. In further embodiments, challenging cows with a microbialcolonizer or microbial pathogen after administering one or morecompositions of the present disclosure prevents the microbial colonizeror microbial pathogen from colonizing cows.

In some embodiments, clearance of the microbial colonizer or microbialpathogen occurs in less than 25 days, less than 24 days, less than 23days, less than 22 days, less than 21 days, less than 20 days, less than19 days, less than 18 days, less than 17 days, less than 16 days, lessthan 15 days, less than 14 days, less than 13 days, less than 12 days,less than 11 days, less than 10 days, less than 9 days, less than 8days, less than 7 days, less than 6 days, less than 5 days, less than 4days, less than 3 days, or less than 2 days post administration of theone or more compositions of the present disclosure.

In some embodiments, clearance of the microbial colonizer or microbialpathogen occurs within 1-30 days, 1-25 days, 1-20 day, 1-15 days, 1-10days, 1-5 days, 5-30 days, 5-25 days, 5-20 days, 5-15 days, 5-10 days,10-30 days, 10-25 days, 10-20 days, 10-15 days, 15-30 days, 15-25 days,15-20 days, 20-30 days, 20-25 days, or 25-30 days post administration ofthe one or more compositions of the present disclosure.

Improved Traits

In some aspects, the present disclosure is drawn to administeringmicrobial compositions described herein to ruminants to improve one ormore traits through the modulation of aspects of milk production, milkquantity, milk quality, ruminant digestive chemistry, and efficiency offeed utilization and digestibility.

In some embodiments, improving the quantity of milk fat produced by aruminant is desirable, wherein milk fat includes triglycerides,triacylglycerides, diacylglycerides, monoacylglycerides, phospholipids,cholesterol, glycolipids, and free fatty acids. In further embodiments,free fatty acids include short chain fatty acids (i.e., C4:0, C6:0, andC8:0), medium chain fatty acids (i.e., C10:0, C10:1, C12:0, C14:0,C14:1, and C15:0), and long chain fatty acids (i.e., C16:0, C16:1,C17:0, C17:1, C18:0, C18:1, C18:2, C18:3, and C20:0). In furtherembodiments, it is desirable to achieve an increase in milk fatefficiency, which is measured by the total weight of milk fat produced,divided by the weight of feed ingested. The weight of milk fat producedis calculated from the measured fat percentage multiplied by the weightof milk produced.

In some embodiments, improving the quantity of carbohydrates in milkproduced by a ruminant is desirable, wherein carbohydrates includelactose, glucose, galactose, and oligosaccharides. Tao et al. (2009. J.Dairy Sci. 92:2991-3001) disclose numerous oligosaccharides that may befound in bovine milk.

In some embodiments, improving the quantity of proteins in milk producedby a ruminant, wherein proteins include caseins and whey. In someembodiments, proteins of interest are only those proteins produced inmilk. In other embodiments, proteins of interest are not required to beproduced only in milk. Whey proteins include immunoglobulins, serumalbumin, beta-lactoglobulin, and alpha-lactoglobulin.

In some embodiments, improving the quantity of vitamins in milk producedby a ruminant is desirable. Vitamins found in milk include thefat-soluble vitamins of A, D, E, and K; as well as the B vitamins foundin the aqueous phase of the milk.

In some embodiments, improving the quantity of minerals in milk producedby a ruminant is desirable. Minerals found in milk include iron, zinc,copper, cobalt, magnesium, manganese, molybdenum, calcium, phosphorous,potassium, sodium, chlorine, and citric acid. Trace amounts of thefollowing may be found in milk: aluminum, arsenic, boron, bromine,cadmium, chromium, fluorine, iodine, lead, nickel, selenium, silicon,silver, strontium, and vanadium.

In some embodiments, improving the milk yield and milk volume producedby a ruminant is desirable. In some embodiments, it is further desirableif the increase in milk yield and volume is not accompanied by simply anincrease in solute volume.

In some embodiments improving energy-corrected milk (ECM) is desirable.In further embodiments, improving ECM amounts to increasing thecalculated ECM output. In some embodiments, the ECM is calculated asfollows: ECM=(0.327×milk pounds)+(12.95×fat pounds)+(7.2×proteinpounds).

In some embodiments, improving the efficiency and digestibility ofanimal feed is desirable. In some embodiments, increasing thedegradation of lignocellulosic components from animal feed is desirable.Lignocellulosic components include lignin, cellulose, and hemicellulose.

In some embodiments, increasing the concentration of fatty acids in therumen of ruminants is desirable. Fatty acids include acetic acid,propionic acid, and butyric acid. In some embodiments, maintaining thepH balance in the rumen to prevent lysis of beneficial microbialconsortia is desirable. In some embodiments, maintaining the pH balancein the rumen to prevent a reduction of beneficial microbial consortia isdesirable.

In some embodiments, decreasing the amount of methane and manureproduced by ruminants is desirable.

In some embodiments, improving the dry matter intake is desirable. Insome embodiments, improving the efficiency of nitrogen utilization ofthe feed and dry matter ingested by ruminants is desirable.

In some embodiments, the improved traits of the present disclosure arethe result of the administration of the presently described microbialcompositions. It is thought that the microbial compositions modulate themicrobiome of the ruminants such that the biochemistry of the rumen ischanged in such a way that the ruminal liquid and solid substratum aremore efficiently and more completely degraded into subcomponents andmetabolites than the rumens of ruminants not having been administeredmicrobial compositions of the present disclosure.

In some embodiments, the increase in efficiency and the increase ofdegradation of the ruminal substratum result in an increase in improvedtraits of the present disclosure.

Mode of Action: Digestibility Improvement in Ruminants

The rumen is a specialized stomach dedicated to the digestion of feedcomponents in ruminants. A diverse microbial population inhabits therumen, where their primary function revolves around converting thefibrous and non-fibrous carbohydrate components into useable sources ofenergy and protein (FIG. 16). Cellulose, in particular, forms up to 40%of plant biomass and is considered indigestible by mammals. It also istightly associated with other structural carbohydrates, includinghemicellulose, pectin, and lignin. The cellulolytic microbes in therumen leverage extensive enzymatic activity in order break thesemolecules down into simple sugars and volatile fatty acids. Thisenzymatic activity is critical to the extraction of energy from feed,and more efficient degradation ultimately provides more energy to theanimal. The soluble sugars found in the non-fibrous portion of the feedare also fermented into gases and volatile fatty acids such as butyrate,propionate, and acetate. Volatile fatty acids arising from the digestionof both the fibrous and non-fibrous components of feed are ultimatelythe main source of energy of the ruminant.

Individual fatty acids have been tested in ruminants in order toidentify their impacts on varying aspects of production.

Acetate: Structural carbohydrates produce large amounts of acetate whendegraded. An infusion of acetate directly into the rumen was shown toimprove the yield of milk, as well as the amount of milk fat produced.Acetate represents at least 90% of acids in the peripheral blood—it ispossible that acetate can be directly utilized by mammary tissue as asource of energy. See Rook and Balch. 1961. Brit. J. Nutr. 15:361-369.

Propionate: Propionate has been shown to increase milk proteinproduction, but decrease milk yield. See Rook and Balch. 1961. Brit. J.Nutr. 15:361-369.

Butyrate: An infusion of butyrate directly into the rumen of dairy cowsincreases milk fat production without changing milk yield. See Huhtanenet al. 1993. J. Dairy Sci. 76:1114-1124.

Network Analysis

A network and/or cluster analysis method, in one embodiment, is used tomeasure connectivity of the one or more strains within a network,wherein the network is a collection of two or more samples that share acommon or similar environmental parameter. In one embodiment, networkanalysis comprises linkage analysis, modularity analysis, robustnessmeasures, betweenness measures, connectivity measures, transitivitymeasures, centrality measures or a combination thereof. In anotherembodiment, network analysis comprises predictive modeling of networkthrough link mining and prediction, social network theory, collectiveclassification, link-based clustering, relational similarity, or acombination thereof. In another embodiment, network analysis comprisesmutual information, maximal information coefficient (MIC) calculations,or other nonparametric methods between variables to establishconnectivity. In another embodiment, network analysis comprisesdifferential equation based modeling of populations. In yet anotherembodiment, network analysis comprises Lotka-Volterra modeling.

The environmental parameter can be a parameter of the sample itself,e.g., pH, temperature, amount of protein in the sample. Alternatively,the environmental parameter is a parameter that affects a change in theidentity of a microbial community (i.e., where the “identity” of amicrobial community is characterized by the type of microorganismstrains and/or number of particular microorganism strains in acommunity), or is affected by a change in the identity of a microbialcommunity. For example, an environmental parameter in one embodiment, isthe food intake of an animal or the amount of milk (or the protein orfat content of the milk) produced by a lactating ruminant In oneembodiment, the environmental parameter is the presence, activity and/orabundance of a second microorganism strain in the microbial community,present in the same sample. In some embodiments, an environmentalparameter is referred to as a metadata parameter.

Other examples of metadata parameters include but are not limited togenetic information from the host from which the sample was obtained(e.g., DNA mutation information), sample pH, sample temperature,expression of a particular protein or mRNA, nutrient conditions (e.g.,level and/or identity of one or more nutrients) of the surroundingenvironment/ecosystem), susceptibility or resistance to disease, onsetor progression of disease, susceptibility or resistance of the sample totoxins, efficacy of xenobiotic compounds (pharmaceutical drugs),biosynthesis of natural products, or a combination thereof.

EXAMPLES Example I Increase Total Milk Fat, Milk Protein, andEnergy-Corrected Milk (ECM) in Cows

The methods of Example I aim to increase the total amount of milk fatand milk protein produced by a lactating ruminant, and the calculatedECM.

The methodologies presented herein—based upon utilizing the disclosedisolated microbes, consortia, and compositions comprising thesame—demonstrate an increase in the total amount of milk fat and milkprotein produced by a lactating ruminant. These increases were realizedwithout the need for further addition of hormones.

In this example, a microbial consortium comprising two isolatedmicrobes, Ascusb_3138 (SEQ ID NO:28; deposited as NRRL Y-67248) andAscusf_15 (SEQ ID NO:32; deposited as NRRL Y67249), was administered toHolstein cows in mid-stage lactation over a period of five weeks.

The cows were randomly assigned into 2 groups of 8, wherein one of thegroups was a control group that received a buffer lacking a microbialconsortium. The second group, the experimental group, was administered amicrobial consortium comprising Ascusb_3138 (SEQ ID NO:28) and Ascusf_15(SEQ ID NO:32) once per day for five weeks. Each of the cows were housedin individual pens and were given free access to feed and water. Thediet was a high milk yield diet. Cows were fed ad libitum and the feedwas weighed at the end of the day, and prior day refusals were weighedand discarded. Weighing was performed with a PS-2000 scale from SalterBrecknell (Fairmont, Minn.).

Cows were cannulated such that a cannula extended into the rumen of thecows. Cows were further provided at least 10 days of recovery postcannulation prior to administering control dosages or experimentaldosages.

Each administration consisted of 20 ml of a neutral buffered saline, andeach administration consisted of approximately 10⁹ cells suspended inthe saline. The control group received 20 ml of the saline once per day,while the experimental group received 20 ml of the saline furthercomprising 10⁹ microbial cells of the described microbial consortium.

The rumen of every cow was sampled on days 0, 7, 14, 21, and 35, whereinday 0 was the day prior to microbial administration. Note that theexperimental and control administrations were performed after the rumenwas sampled on that day. Daily sampling of the rumen, beginning on day0, with a pH meter from Hanna Instruments (Woonsocket, R.I.) wasinserted into the collected rumen fluid for recordings. Rumen samplingincluded both particulate and fluid sampling from the center, dorsal,ventral, anterior, and posterior regions of the rumen through thecannula, and all five samples were pooled into 15 ml conical vialscontaining 1.5 ml of stop solution (95% ethanol, 5% phenol). A fecalsample was also collected on each sampling day, wherein feces werecollected from the rectum with the use of a palpation sleeve. Cows wereweighed at the time of each sampling.

Fecal samples were placed in a 2 ounce vial, stored frozen, and analyzedto determine values for apparent neutral detergent fibers (NDF)digestibility, apparent starch digestibility, and apparent proteindigestibility. Rumen sampling consisted of sampling both fluid andparticulate portions of the rumen, each of which was stored in a 15 mlconical tube. Cells were fixed with a 10% stop solution (5% phenol/95%ethanol mixture) and kept at 4° C. and shipped to Ascus Biosciences(Vista, Calif.) on ice.

The milk yield was measured twice per day, once in the morning and onceat night. Milk composition (% fats and % proteins, etc.) was measuredtwice per day, once in the morning and once at night. Milk samples werefurther analyzed with near-infrared spectroscopy for protein fats,solids, analysis for milk urea nitrogen (MUN), and somatic cell counts(SCC) at the Tulare Dairy Herd Improvement Association (DHIA) (Tulare,Calif.). Feed intake of individual cows and rumen pH were determinedonce per day.

A sample of the total mixed ration (TMR) was collected the final day ofthe adaptation period, and then successively collected once per week.Sampling was performed with the quartering method, wherein the sampleswere stored in vacuum sealed bags which were shipped to CumberlandValley Analytical Services (Hagerstown, Md.) and analyzed with the NIR1package.

The final day of administration of buffer and/or microbial bioconsortiawas on day 35, however all other measurements and samplings continued asdescribed until day 46.

TABLE 12 Dry matter intake, milk production and composition, body weight(BW) gain, and rumen pH least square means (±SEM) of cows assigned toControl and Inoculated groups. Treatment Control Inoculated Outcome Drymatter intake, kg 26.2 ± 2.8  30.2 ± 1.2  Milk yield, kg 25.7 ± 1.9 30.6 ± 1.9  FCM, kg 27.7 ± 2.5  32.5 ± 2.5  ECM, kg 27.2 ± 2.4  32.1 ±2.4  Milk components, % Crude Protein 3.08 ± 0.06 3.27 ± 0.11 Fat 3.87 ±0.08 4.06 ± 0.08 Lactose 4.64 ± 0.10 4.73 ± 0.03 Milk components yield,kg Crude Protein 0.80 ± 0.07 0.97 ± 0.07 Fat 1.01 ± 0.10 1.20 ± 0.10MUN, mg/dL 6.17 ± 0.60 7.41 ± 0.45 FCM/DMI 1.22 ± 0.07 1.10 ± 0.07 Bodyweight gain, kg/day 0.78 ± 0.44 1.46 ± 0.43 Rumen pH 6.24 ± 0.09 6.05 ±0.09

Table 12 reveals the effects of daily administration of an Ascusmicrobial consortium on the performance of multiparous Holstein cows(between 60 and 120 days in milk). Marked differences between thecontrol and inoculated treatments were observed. The inoculated groupexperienced increases in all parameters except FCM/DMI and rumen pH. Theweekly values at the beginning of the intervention period when cows werestill adapting to the treatment are included in the calculations.

FIGS. 4-7 demonstrate the significant effects of the microbial consortiaon dairy cows for daily milk yield, daily milk crude protein yield,daily milk fat yield, and daily energy corrected milk yield over time.After an initial adaptation period, during which the microbes wereobserved to colonize the rumen, the production characteristics of theinoculated treatment group increased and diverged from the controlgroup.

FIG. 3A demonstrates that cows that were administered the microbialconsortia exhibited a 20.9% increase in the average production of milkfat versus cows that were administered the buffered solution alone. FIG.3B demonstrates that cows that were administered the microbial consortiaexhibited a 20.7% increase in the average production of milk proteinversus cows that were administered the buffered solution alone. FIG. 3Cdemonstrates that cows that were administered the microbial consortiaexhibited a 19.4% increase in the average production of energy correctedmilk. The increases seen in FIG. 3A-C became less pronounced after theadministration of the consortia ceased, as depicted by the vertical lineintersecting the data points.

FIG. 14 clearly identifies the effect of microbial consortia on thesomatic cell count in the milk. The experimental group of cows receivingthe microbial consortia exhibited a decrease in the number of cows withgreater than 200,000 somatic cells/ml of milk. In the field of dairyfarming, the SCC is a strong indicator of milk quality. The majority ofsomatic cells found in milk are leukocytes, immune cells that accumulatein a particular tissue/fluid in increasing numbers usually due to animmune response to a pathogen. Generally, the lower the SCC the higherthe quality of milk. Dosogne et al. 2011. J. Dairy Sci. 86(3):828-834.

Example II Increase Total Milk Fat and Milk Protein in Cows

In certain embodiments of the disclosure, the present methods aim toincrease the total amount of milk fat and milk protein produced by alactating ruminant.

The methodologies presented herein—based upon utilizing the disclosedisolated microbes, consortia, and compositions comprising the same—havethe potential to increase the total amount of milk fat and milk proteinproduced by a lactating ruminant. These increases can be realizedwithout the need for further addition of hormones.

In this example, seven microbial consortia comprising isolated microbesfrom Table 1 are administered to Holstein cows in mid-stage lactationover a period of six weeks. The consortia are as follows:

Consortium 1—Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_24;

Consortium 2—Ascusb_7, Ascusb_1801, Ascusf_45, and Ascusf_24;

Consortium 3—Ascusb_7, Ascusb_268, Ascusf_45, and Ascusf_24;

Consortium 4—Ascusb_7, Ascusb_232, Ascusf_45, and Ascusf_24;

Consortium 5—Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_249;

Consortium 6—Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_353; and

Consortium 7—Ascusb_7, Ascusb_32, Ascusf_45, and Ascusf_23.

Consortium 8—Ascusb_3138, Ascusb_1801, Ascusf_45, and Ascusf_15.

Consortium 9—Ascusb_3138, Ascusb_268, Ascusf_45, and Ascusf_15.

Consortium 10—Ascusb_3138, Ascusb_232, Ascusf_23, and Ascusf_15.

Consortium 11—Ascusb_7, Ascusb_3138, Ascusf_15, and Ascusf_249.

Consortium 12—Ascusb_7, Ascusb_3138, Ascusf_45, and Ascusf_15.

Consortium 13—Ascusb_3138, Ascusb_32, Ascusf_15, and Ascusf_23.

Consortium 14—Ascusb_3138 and Ascusf_15.

The cows are randomly assigned into 15 groups of 8, wherein one of thegroups is a control group that receives a buffer lacking a microbialconsortium. The remaining seven groups are experimental groups and willeach be administered one of the thirteen microbial bioconsortia once perday for six weeks. Each of the cows are held in individual pens tomitigate cross-contamination and are given free access to feed andwater. The diet is a high milk yield diet. Cows are fed twice per dayand the feed will be weighed at each feeding, and prior day refusalswill be weighed and discarded. Weighing is performed with a PS-2000scale from Salter Brecknell (Fairmont, Minn.).

Cows are cannulated such that a cannula extends into the rumen of thecows. Cows are further provided at least 10 days of recovery postcannulation prior to administering control dosages or experimentaldosages.

Each administration consists of 5 ml of a neutral buffered saline, andeach administration consists of approximately 10⁹ cells suspended in thesaline. The control group receives 5 ml of the saline once per day,while the experimental groups receive 5 ml of the saline furthercomprising 10⁹ microbial cells of the described consortia.

The rumen of every cow is sampled on days 0, 7, 14, 21, and 35, whereinday 0 is the day prior to microbial administration. Note that theexperimental and control administrations are performed after the rumenhas been sampled on that day. Daily sampling of the rumen, beginning onday 0, with a pH meter from Hanna Instruments (Woonsocket, R.I.) isinserted into the collected rumen fluid for recordings. Rumen samplingincluded both particulate and fluid sampling from the center, dorsal,ventral, anterior, and posterior regions of the rumen through thecannula, and all five samples were pooled into 15 ml conical vialscontaining 1.5 ml of stop solution (95% ethanol, 5% phenol). A fecalsample is also collected on each sampling day, wherein feces arecollected from the rectum with the use of a palpation sleeve. Cows areweighed at the time of each sampling.

Fecal samples are placed in a 2 ounce vial, stored frozen, and analyzedto determine values for apparent NDF digestibility, apparent starchdigestibility, and apparent protein digestibility. Rumen samplingconsists of sampling both fluid and particulate portions of the rumen,each of which is stored in a 15 ml conical tube. Cells are fixed with a10% stop solution (5% phenol/95% ethanol mixture) and kept at 4° C. andshipped to Ascus Biosciences (Vista, Calif.) on ice.

The milk yield is measured twice per day, once in the morning and onceat night. Milk composition (% fats and % proteins, etc.) is measuredtwice per day, once in the morning and once at night. Milk samples arefurther analyzed with near-infrared spectroscopy for protein fats,solids, analysis for milk urea nitrogen (MUN), and somatic cell counts(SCC) at the Tulare Dairy Herd Improvement Association (DHIA) (Tulare,Calif.). Feed intake of individual cows and rumen pH are determined onceper day.

A sample of the total mixed ration (TMR) is collected the final day ofthe adaptation period, and then successively collected once per week.Sampling is performed with the quartering method, wherein the samplesare stored in vacuum sealed bags which are shipped to Cumberland ValleyAnalytical Services (Hagerstown, Md.) and analyzed with the NIR1package.

In some embodiments, the percent fats and percent protein of milk ineach of the experimental cow groups is expected to demonstrate astatistically significant increase over the percent fats and percentprotein of milk in the control cow group. In other embodiments, theincrease is not expected to be statistically significant, but it isexpected to be still quantifiable.

Example III Shifting the Microbiome of Ruminants

In certain embodiments of the disclosure, the present methods aim tomodulate the microbiome of ruminants through the administration of oneor more microbes to the gastrointestinal tract of ruminants.

The methodologies presented herein—based upon utilizing the disclosedisolated microbes, consortia, and compositions comprising the same—havethe potential to modulate the microbiome of ruminants. The modulation ofa ruminant's gastrointestinal microbiome may lead to an increase ofdesirable traits of the present disclosure.

In this example, the microbial consortia of Table 5 are administered toHolstein cows over a period of six weeks.

The cows are randomly assigned into 37 groups of 8, wherein one of thegroups is a control group that receives a buffer lacking a microbialconsortium. The remaining thirty-six groups are experimental groups andwill each be administered one of the thirty-six microbial consortia onceper day for six weeks. Each of the cows are held in individual pens tomitigate cross-contamination and are given free access to feed andwater. The diet is a high milk yield diet. Cows are fed twice per dayand the feed will be weighed at each feeding, and prior day refusalswill be weighed and discarded. Weighing is performed with a PS-2000scale from Salter Brecknell (Fairmont, Minn.).

Cows are cannulated such that a cannula extends into the rumen of thecows. Cows are further provided at least 10 days of recovery postcannulation prior to administering control dosages or experimentaldosages.

Each administration consists of 5 ml of a neutral buffered saline, andeach administration consists of approximately 10⁹ cells suspended in thesaline. The control group receives 5 ml of the saline once per day,while the experimental groups receive 5 ml of the saline furthercomprising 10⁹ microbial cells of the described consortia.

The rumen of every cow is sampled on days 0, 7, 14, 21, and 35, whereinday 0 is the day prior to administration. Note that the experimental andcontrol administrations are performed after the rumen has been sampledon that day. Daily sampling of the rumen, beginning on day 0, with a pHmeter from Hanna Instruments (Woonsocket, R.I.) is inserted into thecollected rumen fluid for recordings. Rumen sampling included bothparticulate and fluid sampling from the center, dorsal, ventral,anterior, and posterior regions of the rumen through the cannula, andall five samples were pooled into 15 ml conical vials containing 1.5 mlof stop solution (95% ethanol, 5% phenol). A fecal sample is alsocollected on each sampling day, wherein feces are collected from therectum with the use of a palpation sleeve. Cows are weighed at the timeof each sampling.

Fecal samples are placed in a 2 ounce vial, stored frozen, and analyzedto determine values for apparent NDF digestibility, apparent starchdigestibility, and apparent protein digestibility. Rumen samplingconsists of sampling both fluid and particulate portions of the rumen,each of which is stored in a 15 ml conical tube. Cells are fixed with a10% stop solution (5% phenol/95% ethanol mixture) and kept at 4° C. andshipped to Ascus Biosciences (Vista, Calif.) on ice.

The samples of fluid and particulate portions of the rumen, as well asthe fecal samples are each evaluated for microbiome fingerprintingutilizing the T-RFLP method combined with nMDS ordination and PERMANOVAstatistics.

In some embodiments, the ruminal and fecal microbiome in each of theexperimental cow groups is expected to demonstrate a statisticallysignificant change in the microbiomes over the microbiomes in thecontrol cow group as well as the 0 day microbiome samples, wherein thechange is a significant increase in the proportion of microbesadministered in the experimental administrations. In other embodiments,the increase is not expected to be statistically significant, but it isexpected to be still quantifiable.

Example IV Milk Fat Produced Versus Absolute Abundance of Microbes

Determine rumen microbial community constituents that impact theproduction of milk fat in dairy cows.

Eight lactating, ruminally cannulated, Holstein cows were housed inindividual tie-stalls for use in the experiment. Cows were fed twicedaily, milked twice a day, and had continuous access to fresh water. Onecow (cow 4201) was removed from the study after the first dietary MilkFat Depression due to complications arising from an abortion prior tothe experiment.

Experimental Design and Treatment: The experiment used a crossoverdesign with 2 groups and 1 experimental period. The experimental periodlasted 38 days: 10 days for the covariate/wash-out period and 28 daysfor data collection and sampling. The data collection period consistedof 10 days of dietary Milk Fat Depression (MFD) and 18 days of recovery.After the first experimental period, all cows underwent a 10-day washout period prior to the beginning of period 2.

Dietary MFD was induced with a total mixed ration (TMR) low in fiber(29% NDF) with high starch degradability (70% degradable) and highpolyunsaturated fatty acid levels (PUFA, 3.7%). The Recovery phaseincluded two diets variable in starch degradability. Four cows wererandomly assigned to the recovery diet high in fiber (37% NDF), low inPUFA (2.6%), and high in starch degradability (70% degradable). Theremaining four cows were fed a recovery diet high in fiber (37% NDF),low in PUFA (2.6%), but low in starch degradability (35%).

During the 10-day covariate and 10-day wash out periods, cows were fedthe high fiber, low PUFA, and low starch degradability diet.

Samples and Measurements: Milk yield, dry matter intake, and feedefficiency were measured daily for each animal throughout the covariate,wash out, and sample collection periods. TMR samples were measured fornutrient composition. During the collection period, milk samples werecollected and analyzed every 3 days. Samples were analyzed for milkcomponent concentrations (milk fat, milk protein, lactose, milk ureanitrogen, somatic cell counts, and solids) and fatty acid compositions.

Rumen samples were collected and analyzed for microbial communitycomposition and activity every 3 days during the collection period. Therumen was intensively sampled 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and22 hours after feeding during day 0, day 7, and day 10 of the dietaryMFD. Similarly, the rumen was intensively sampled 0, 2, 4, 6, 8, 10, 12,14, 16, 18, 20, and 22 hours after feeding on day 16 and day 28 of thesample collection period. Rumen contents were analyzed for pH, acetateconcentration, butyrate concentration, propionate concentration, isoacidconcentration, and long chain and CLA isomer concentrations. Rumensampling included both particulate and fluid sampling from the center,dorsal, ventral, anterior, and posterior regions of the rumen throughthe cannula, and all five samples were pooled into 15 ml conical vials.

Rumen Sample Preparation and Sequencing: After collection, rumen sampleswere centrifuged at 4,000 rpm in a swing bucket centrifuge for 20minutes at 4° C. The supernatant was decanted, and an aliquot of eachrumen content sample (1-2 mg) was added to a sterile 1.7 mL tubeprefilled with 0.1 mm glass beads. A second aliquot was collected andstored in an empty, sterile 1.7 mL tube for cell counting.

Rumen samples in empty tubes were stained and put through a flowcytometer to quantify the number of cells of each microorganism type ineach sample. Rumen samples with glass beads were homogenized with beadbeating to lyse microorganisms. DNA and RNA was extracted and purifiedfrom each sample and prepared for sequencing on an Illumina Miseq.Samples were sequenced using paired-end chemistry, with 300 base pairssequenced on each end of the library.

Sequencing Read Processing and Data Analysis: Sequencing reads werequality trimmed and processed to identify bacterial species present inthe rumen based on a marker gene, 16S rDNA, or ITS1 and/or ITS2. Countdatasets and activity datasets were integrated with the sequencing readsto determine the absolute cell numbers of active microbial specieswithin the rumen microbial community. Production characteristics of thecow over time, including pounds of milk produced, were linked to thedistribution of active microorganisms within each sample over the courseof the experiment using mutual information.

Tests cases to determine the impact of count data, activity data, andcount and activity on the final output were run by omitting theappropriate datasets from the sequencing analysis. To assess the impactof using a linear correlation rather than the MIC on target selection,Pearson's coefficients were also calculated for pounds of milk fatproduced as compared to the relative abundance of all microorganisms andthe absolute abundance of active microorganisms.

Results

One component of the Ascus Biosciences technology utilized in thisapplication leverages mutual information to rank the importantce ofnative microbial strains residing in the gastrointestinal tract of theanimal to specific animal traits. The maximal information coefficient(MIC) scores are calculated for all microorganisms and the desiredanimal trait. Relationships were scored on a scale of 0 to 1, with 1represending a strong relationship between the microbial strain and theanimal trait, and 0 representing no relationship. A cut-off based onthis score is used to define useful and non-useful microorganisms withrespect to the improvement of specific traits. FIG. 9 and FIG. 10 depictthe MIC score distribution for rumen microbial strains that share arelationship with milk fat efficiency in dairy cows. The point where thecurve shifts from exponential to linear (˜0.45-0.5 for bacteria and ˜0.3for fungi) represents the cutoff between useful and non-usefulmicroorganism strains pertaining to milk fat efficiency. FIG. 11 andFIG. 12 depict the MIC score distributions for rumen microbial strainsthat share a relationship with dairy efficiency. The point where thecurve shifts from exponential to linear (˜0.45-0.5 for bacteria and˜0.25 for fungi) represents the cutoff between useful and non-usefulmicroorganism strains.

The MICs were calculated between pounds of milk fat produced and theabsolute abundance of each active microorganism. Microorganisms wereranked by MIC score, and microorganisms with the highest MIC scores wereselected as the target species most relevant to pounds of milk produced.MIC scores of the microbes of the present disclosure are recited inTable 1. The greater the MIC score, the greater the ability of themicrobe to confer an increase in the weight of milk fat produced by acow

Example V Comparative Analysis of MIC Scores From Published Work ofOther Groups

Utilizing Ascus Biosciences' technology, the performance of currentlyavailable microbial feed additive products was predicted.

Direct-fed microbial products that claim to enhance dairy performanceare openly available on the market. Some of these products containmicroorganism strains that are native rumen microorganisms (Megasphaeraelsdenii), or are within 97% sequence similarity of native rumenmicroorganisms. We have identified the species of microbes utilized inthese products, and calculated their MIC score with respect to milk fatefficiency (FIG. 13). As evidenced by the curve in FIG. 13, all of theassayed strains that were available fell below the threshold used todefine useful and non-useful strains, as describe above. Thespecies/strain closest to the cutoff, Prevotella bryantii, has shown apositive effect in one study.

Lactobacillus plantarum: MIC 0.28402

The calculated MIC predicts that Lactobacillus plantarum is poorlyassociated with milk fat efficiency, and the art discloses that aninoculation of L. plantarum yields no increase in milk fat product, andat least one study discloses that some strains of L. plantarum createmolecules that cause milk fat depression. See Lee et al. 2007. J. Appl.Microbiol. 103(4):1140-1146 and Mohammed et al. 2012. J. Dairy Sci.95(1):328-339.

Lactobacillus acidophilus: MIC 0.30048

The calculated MIC predicts that Lactobacillus acidophilus is poorlyassociated with milk fat efficiency, and the art discloses that theadministration of L. acidophilus to dairy cows/calves had no effect ofvarious aspects of milk yield/milk component yield. See Higginbotham andBath. 1993. J. Dairy Sci. 76(2):615-620; Abu-Tarboush et al. 1996.Animal Feed Sci. Tech. 57(1-2):39-49; McGilliard and Stallings. 1998. J.Dairy Sci. 81(5):1353-1357; and Raeth-Knight et al. 2007. J. Dairy Sci.90(4):1802-1809; But see Boyd et al. 2011. 94(9):4616-4622 (discloses anincrease in milk yield and milk protein yield).While Boyd et al. doesdisclose an increase in milk and milk protein yield, the controls ofthis single study do not appear to adequately isolate the the presenceof L. acidophilus as the cause of the increase. The body of prior artcontradicts the finding of Boyd et al.

Megasphaera elsdenii: MIC 0.32548

The calculated MIC predicts that Megasphaera elsdenii is poorlyassociated with milk fat efficiency, and the art provides substantialevidence to suggest that Megasphaera elsdenii has no positive effectupon milk fat efficiency, but multiple references provide evidence tosuggest that it has a negative effect on milk fat efficiency. See Kim etal. 2002. J. Appl. Micro. 92(5):976-982; Hagg. 2008. Dissertation,University of Pretoria. 1-72; Hagg et al. 2010. S. African. J. AnimalSci. 40(2):101-112; Zebeli et al. 2011. J. Dairy Res. 79(1):16-25;Aikman et al. 2011. J. Dairy Sci. 94(6):2840-2849; Mohammed et al. 2012.J. Dairy Sci. 95(1):328-339; and Cacite and Weimer. 2016. J. Animal Sci.Poster Abstract. 94(sup. 5):784.

Prevotella bryantii: MIC 0.40161

The calculated MIC predicts that Prevotella bryantii is not highlyassociated with milk fat efficiency, and the art provides evidence thatP. bryantii administered during subacute acidosis challenge inmidlactation dairy cows has no apparent effect on milk yield, whereasadministration of the microbe to dairy cows in early lactation yieldsimproved milk fat concentrations. See Chiquette et al. 2012. J. DairySci. 95(10):5985-5995, but see Chiquette et al. 2008. 91(9):3536-3543;respectively.

Example VI Shift in Rumen Microbial Composition after Administration ofa Microbial Composition

The methods of the instant example aim to increase the total amount ofmilk fat and milk protein produced by a lactating ruminant, and thecalculated energy corrected milk (ECM).

The methodologies presented herein—based upon utilizing the disclosedisolated microbes, consortia, and compositions comprising thesame—demonstrate an increase in the total amount of milk fat and milkprotein produced by a lactating ruminant. These increases were realizedwithout the need for further addition of hormones.

In this example, a microbial consortium comprising two isolatedmicrobes, Ascusb_3138 (SEQ ID NO:28) and Ascusf_15 (SEQ ID NO:32), wasadministered to Holstein cows in mid-stage lactation over a period offive weeks.

The cows were randomly assigned into 2 groups of 8, in which one of thegroups was a control group that received a buffer lacking a microbialconsortium. The second group, the experimental group, was administered amicrobial consortium comprising Ascusb_3138 (SEQ ID NO:28) and Ascusf_15(SEQ ID NO:32) once per day for five weeks. Each cow was housed in anindividual pen and was given free access to feed and water. The diet wasa high milk yield diet. Cows were fed ad libitum and the feed wasweighed at the end of each day, and prior day refusals were weighed anddiscarded. Weighing was performed with a PS-2000 scale from SalterBrecknell (Fairmont, Minn.).

Cows were cannulated such that a cannula extended into the rumen of thecows. Cows were further provided at least 10 days of recovery postcannulation prior to administering control dosages or experimentaldosages.

Each administration consisted of 20 ml of a neutral buffered saline, andeach administration consisted of approximately 10⁹ cells suspended inthe saline. The control group received 20 ml of the saline once per day,while the experimental group received 20 ml of the saline furthercomprising 10⁹ microbial cells of the described microbial consortium.

The rumen of every cow was sampled on days 0, 7, 14, 21, and 35, whereinday 0 was the day prior to microbial administration. Note that theexperimental and control administrations were performed after the rumenwas sampled on that day. Daily sampling of the rumen, beginning on day0, with a pH meter from Hanna Instruments (Woonsocket, R.I.) wasinserted into the collected rumen fluid for recordings. Rumen samplingincluded both particulate and fluid sampling from the center, dorsal,ventral, anterior, and posterior regions of the rumen through thecannula, and all five samples were pooled into 15 ml conical vialscontaining 1.5 ml of stop solution (95% ethanol, 5% phenol) and storedat 4° C. and shipped to Ascus Biosciences (Vista, Calif.) on ice.

A portion of each rumen sample was stained and put through a flowcytometer to quantify the number of cells of each microorganism type ineach sample. A separate portion of the same rumen sample was homogenizedwith bead beating to lyse microorganisms. DNA and RNA was extracted andpurified from each sample and prepared for sequencing on an IlluminaMiseq. Samples were sequenced using paired-end chemistry, with 300 basepairs sequenced on each end of the library. The sequencing reads wereused to quantify the number of cells of each active, microbial memberpresent in each animal rumen in the control and experimental groups overthe course of the experiment.

Ascusb_3138 and Ascusf_15 both colonized, and were active in the rumenafter ˜3-5 days of daily administration, depending on the animal. Thiscolonization was observed in the experimental group, but not in thecontrol group. The rumen is a dynamic environment, where the chemistryof the cumulative rumen microbial population is highly intertwined. Theartificial addition of Ascusb_3138 and Ascuf_15 could have effects onthe overall structure of the community. To assess this potential impact,the entire microbial community was analyzed over the course of theexperiment to identify higher level taxonomic shifts in microbialcommunity population.

Distinct trends were not observed in the fungal populations over time,aside from the higher cell numbers of Ascusf_15 in the experimentalanimals. The bacterial populations, however, did change morepredictably. To assess high level trends across individual animals overtime, percent compositions of the microbial populations were calculatedand compared. See

Table 13. Only genera composing greater than 1% of the community wereanalyzed. The percent composition of genera containing knownfiber-degrading bacteria, including Ruminococcus, was found to increasein experimental animals as compared to control animals. Volatile fattyacid-producing genera, including Clostridial cluster XIVa, Clostridium,Pseudobutyrivibrio, Butyricimonas, and Lachnospira were also found athigher abundances in the experimental animals. The greatest shift wasobserved in the genera Prevotella. Members of this genus have been shownto be involved in the digestion of cellobiose, pectin, and various otherstructural carbohydrates within the rumen. Prevotella sp. have furtherbeen implicated in the conversion of plant lignins into beneficialantioxidants (Schogor et al. PLOS One. 9(4):e87949 (10 p.)).

To more directly measure quantitative changes in the rumen over time,cell count data was integrated with sequencing data to identify bulkchanges in the population at the cell level. Fold changes in cellnumbers were determined by dividing the average number of cells of eachgenera in the experimental group by the average number of cells of eachgenera in the control group. See Table 13. The cell count analysiscaptured many genera that fell under the threshold in the previousanalysis Promicromonospora, Rhodopirellula, Olivibacter, Victivallis,Nocardia, Lentisphaera, Eubacteiru, Pedobacter, Butyricimonas,Mogibacterium, and Desulfovibrio were all found to be at least 10 foldhigher on average in the experimental animals. Prevotella, Lachnospira,Butyricicoccus, Clostridium XIVa, Roseburia, Clostridium_sensu_stricto,and Pseudobutyrivibrio were found to be ˜1.5 times higher in theexperimental animals.

TABLE 13 Family and Genus Level Analysis of Shifts in BacterialPopulations Experi- Control Varia- mental Varia- Taxonomy (%) tion (%)tion Family Level Analysis Prevotellaceae 15.27 6.43 18.62 5.63Ruminococcaceae 16.40 5.14 17.84 6.44 Lachnospiraceae 23.85 7.63 24.586.96 Genus Level Analysis Prevotella 16.14 5.98 19.14 5.27Clostridium_XIVa 12.41 5.35 12.83 4.81 Lachnospiracea_incertae_sedis3.68 1.68 3.93 1.33 Ruminococcus 3.70 2.21 3.82 1.82 Clostridium_IV 3.021.87 3.51 1.74 Butyricimonas 1.68 1.35 1.83 2.38Clostridium_sensu_stricto 1.52 0.65 1.81 0.53 Pseudobutyrivibrio 1.000.64 1.42 1.03 Citrobacter 0.71 1.86 1.95 3.00 Selenomonas 1.04 0.831.34 0.86 Hydrogeno- 1.03 1.08 1.11 0.78 anaerobacterium

TABLE 14 Analysis of Fold Changes in Bacterial cells Genus Fold change(experimental/control) Promicromonospora 22619.50 Rhodopirellula 643.31Olivibacter 394.01 Victivallis 83.97 Nocardia 73.81 Lentisphaera 57.70Eubacterium 50.19 Pedobacter 26.15 Butyricimonas 15.47 Mogibacterium15.23 Desulfovibrio 13.55 Anaeroplasma 8.84 Sharpea 8.78Erysipelotrichaceae_incertae_sedis 5.71 Saccharofermentans 5.09Parabacteroides 4.16 Papillibacter 3.63 Citrobacter 2.95Lachnospiracea_incertae_sedis 2.27 Prevotella 1.60 Butyricicoccus 1.95Clostridium_XIVa 1.47 Roseburia 1.44 Pseudobutyrivibrio 1.43Clostridium_sensu_stricto 1.29 Selenomonas 1.25 Olsenella 1.04

Example VII Analysis of Rumen Microbes for Volatile Fatty AcidProduction and Carbon Source Use A. Volatile Fatty Acid (VFA) Production

To assess the ability of the strains to produce volatile fatty acids,High Performance Liquid Chromatography (HPLC) was utilized to measurethe conventration sof acetate, butyrate, and propionate in spent media.M2GSC media was used in an assay mimicking rumen conditions as closelyas possibile.

For pure cultures, a single colony from each of the desired strains(from anaerobic agar plates) was inoculated into M2GSC media. A mediumblank (control) was also prepared. Cultures and the medium blank wereincubated at 37° C. until significant growth was visible. An opticaldensity (OD600) was determined for each culture, and the strain ID wasconfirmed with Illumina sequencing. An aliquot of culture was subjectedto sterile filtration into a washed glass 15 ml sample vial and analyzedby HPLC; HPLC assays were performed at Michigan State University.Enrichments that exhibited growth were also stained and cell counted toconfirm that the individual strains within each enrichment grew. Strainsoften appeared in multiple enrichments, so the enrichment with thehighest amount of growth for the strain (i.e. the highest increase incell number of that strain) is reported in Table 15.

Due to the vast complexity of metabolisms and microbial lifestylespresent in the rumen, many rumen microorganisms are incapable of axenicgrowth. In order to assay these organisms for desirable characteristics,enrichments cultures were established under a variety of conditions thatmimicked particular features of the rumen environment. Diluted rumenfluid ( 1/100 dilution) was inoculated into M2GSC or M2 mediasupplemented with a variety of carbon sources including xylose (4 g/L),mannitol (4 g/L), glycerol (4 g/L), xylan (2 g/L), cellobiose (2 g/L),arabinose (4 g/L), mannose (4 g/L), rhaminose (2 g/L), maltose (2 g/L),maltose (2 g/L), and molasses. Rumen fluid was also sometimes omittedfrom the recipe. Additions including amino acids, volatile fatty acids,and antibiotics, were also varied across the enrichments. A medium blank(control) was also prepared. Cultures and the medium blank wereincubated at 37° C. until significant growth was visible. An opticaldensity (OD600) was determined for each culture, and the strain IDs wereconfirmed with Illumina sequencing. An aliquot of culture was subjectedto sterile filtration into a washed glass 15 ml sample vial and analyzedby HPLC; HPLC assays were performed at Michigan State University.Enrichments that exhibited growth were also stained and cell counted toconfirm that the individual strains within each enrichment grew. Strainsoften appeared in multiple enrichments, so the enrichment with thehighest amount of growth for the strain (i.e, the highest increase incell number of that strain) is reported in Table 15.

Concentrations of acetate, butyrate, and propionate were quantified forthe medium blanks as well as the sterile filtered culture samples forboth pure strain and enrichment experiments. HPLC parameters were asfollows: Biorad Aminex HPX-87H column, 60° C., 0.5 ml/minute mobilephase 0.00325 N H₂SO₄, 500 psi, 35C RI detector, 45 minute run time, and5 μL injection volume. Concentrations of acetate, butyrate, andpropionate for both pure cultures and enrichments are reported in Table15.

TABLE 15 Volatile Fatty Acid Production of Microbial Strains as Analyzedwith HPLC, Normalized to 1 OD Sample ID Acetate (g/L) Propionate (g/L)Butyrate (g/L) Ascusb_5 3.59 0.00 0.00 Ascusb_7 1.54 4.08 0.03 Ascusb_11−6.88 −0.28 −0.04 Ascusb_26 6.10 7.57 1.38 Ascusb_27 0.59 1.48 4.98Ascusb_32 6.10 7.57 1.38 Ascusb_36 4.30 0.68 0.00 Ascusb_79 2.00 0.000.00 Ascusb_82 6.10 7.57 1.38 Ascusb_89 1.69 4.20 0.27 Ascusb_101 1.45−0.21 0.00 Ascusb_102 2.00 0.00 0.00 Ascusb_104 27.13 34.55 3.31Ascusb_111 1.69 4.20 0.27 Ascusb_119 1.54 4.08 0.03 Ascusb_125 10.975.68 4.69 Ascusb_145 1.69 4.20 0.27 Ascusb_149 0.00 0.00 0.47 Ascusb_1597.05 4.52 1.42 Ascusb_183 0.00 0.00 2.03 Ascusb_187 10.97 5.68 4.69Ascusb_190 7.40 7.36 7.91 Ascusb_199 11.36 1.17 7.65 Ascusb_205 6.107.57 1.38 Ascusb_232 7.83 1.15 3.19 Ascusb_268 2.00 0.00 0.00 Ascusb_2787.05 4.52 1.42 Ascusb_329 7.83 1.15 3.19 Ascusb_368 1.69 4.20 0.27Ascusb_374 7.83 1.15 3.19 Ascusb_411 1.69 4.20 0.27 Ascusb_546 4.30 0.680.00 Ascusb_728 2.36 0.00 0.00 Ascusb_765 −11.63 0.00 0.00 Ascusb_8101.54 4.08 0.03 Ascusb_812 2.00 0.00 0.00 Ascusb_817 1.16 0.00 0.09Ascusb_826 0.42 0.00 0.51 Ascusb_880 −0.12 0.00 0.00 Ascusb_913 10.975.68 4.69 Ascusb_974 4.30 0.68 0.00 Ascusb_1069 0.00 0.00 2.32Ascusb_1074 7.05 4.52 1.42 Ascusb_1295 1.54 4.08 0.03 Ascusb_1367 7.407.36 7.91 Ascusb_1632 1.54 4.08 0.03 Ascusb_1674 0.68 0.30 0.00Ascusb_1763 1.69 4.20 0.27 Ascusb_1780 1.32 0.00 0.21 Ascusb_1786 1.694.20 0.27 Ascusb_1801 5.47 26.95 −0.60 Ascusb_1812 1.54 4.08 0.03Ascusb_1833 7.83 1.15 3.19 Ascusb_1850 1.32 0.00 0.21 Ascusb_2090 1.544.08 0.03 Ascusb_2124 1.69 4.20 0.27 Ascusb_2511 0.00 0.00 0.11Ascusb_2530 11.36 1.17 7.65 Ascusb_2597 4.30 0.68 0.00 Ascusb_2624 0.000.00 0.00 Ascusb_2667 3.16 1.46 1.02 Ascusb_2836 1.32 0.00 0.21Ascusb_3003 0.00 0.00 0.11 Ascusb_3138 0.00 0.00 2.50 Ascusb_3504 1.694.20 0.27 Ascusb_3881 7.05 4.52 1.42 Ascusb_6589 5.47 26.95 −0.60Ascusb_12103 0.94 0.00 0.00 Ascusb_14245 1.76 0.00 0.00 Ascusb_2008327.13 34.55 3.31 Ascusb_20187 7.40 7.36 7.91

B. Soluble Carbon Source Assay

To assess the ability of the strains to degrade various carbon sources,an optical density (OD600) was used to measure growth of strains onmultiple carbon sources over time.

For pure isolates, a single colony from each of the desired strains(from anaerobic agar plates) was inoculated into M2GSC media. A mediumblank (control) was also prepared. Strains were inoculated into a carbonsource assay anaerobically, wherein the assay was set up in a 2 mLsterile 96-well plate, with each well containing RAMM salts, vitamins,minerals, cysteine, and a single carbon source. Carbon sources includedglucose, xylan, lactate, xylose, mannose, glycerol, pectin, molasses,and cellobiose. Cells were inoculated such that each well started at anOD600 of 0.01. Optican densities were read at 600 nm with the Synergy H4hybrid plate reader. The strain IDs were confirmed with Illuminasequencing after all wells were in stationary phase.

As in the volatile fatty acid assay above, enrichments were also used toassay carbon source degradation. Diluted rumen fluid ( 1/100 dilution)was inoculated into M2GSC or M2 media supplemented with a variety ofcarbon sources including xylose (4 g/L), mannitol (4 g/L), glycerol (4g/L), xylan (2 g/L), cellobiose (2 g/L), arabinose (4 g/L), mannose (4g/L), rhaminose (2 g/L), maltose (2 g/L), maltose (2 g/L), and molasses.Rumen fluid was also sometimes omitted from the recipe. Additionsincluding amino acids, volatile fatty acids, and antibiotics, were alsovaried across the enrichments. A medium blank (control) was alsoprepared. Cultures and the medium blank were incubated at 37° C. untilsignificant growth was visible. An optical density (OD600) wasdetermined for each culture, and the strain IDs were confirmed withIllumina sequencing. Enrichments that exhibited growth were also stainedand cell counted to confirm that the individual strains within eachenrichment grew.

C. Insoluble Carbon Source Assay

To assess the ability of the strains to degrade insoluble carbonsources, visual inspection was leveraged to qualitatively determine astrain's degradation capabilities.

For pure cultures, a single colony from each of the desired strains(from anaerobic agar plates) was inoculated into anaerobic Hungate tubescontaining Lowe's semi defined media with cellulose paper, starch, orgrass as the sole carbon source. (Lowe et al. 1985. J. Gen. Microbiol.131:2225-2229). Enrichment cultures using a 1/100 dilutiopn of rumenfluid were also set up using the same medium conditions. Cultures werechecked visually for degradation of insoluble carbon sources (See FIG.14). Strain ID was confirmed using Illumina sequencing. Enrichments thatexhibited growth were also stained and cell counted to confirm that theindividual strains within each enrichment grew.

TABLE 16 Analysis of Degradation of Various Soluable and Non-SoluableCarbon Sources by Strains of the Present Disclosure Strain ID D-GlucoseXylan Lactate D-Xylose D-Mannose Glycerol Pectin Molasses CellobioseCellulose Starch Ascusb_5 + + − + + + + − + Unknown Unknown Ascusb_7 +− + − − + − − + Unknown Unknown Ascusb_11 − − − + − + + + + UnknownUnknown Ascusb_26 + − + − − + − − + Unknown Unknown Ascusb_27 + − − − −− − − − Unknown Unknown Ascusb_32 + − + − + + − − + Unknown UnknownAscusb_36 − + − − − + − − − Unknown Unknown Ascusb_79 + − − − − + − − +Unknown Unknown Ascusb_82 + + + + − + − − + Unknown Unknown Ascusb_89 +− − − − + − − − Unknown Unknown Ascusb_101 − − + − − + − − − UnknownUnknown Ascusb_102 + + + − − + − − − Unknown Unknown Ascusb_104 − − + −− − − − − Unknown Unknown Ascusb_111 − + + − − + − − + Unknown UnknownAscusb_119 − − − + − + − − − Unknown Unknown Ascusb_125 − − + + − + − +− Unknown Unknown Ascusb_145 + − − − − + − − − Unknown UnknownAscusb_149 + − − + + + − + − Unknown Unknown Ascusb_159 + − + + − + − +− Unknown Unknown Ascusb_183 + − − + − + − + + Unknown UnknownAscusb_187 + + − + − + − + − Unknown Unknown Ascusb_190 + − + − − + − +− Unknown Unknown Ascusb_199 − − − + − + − − − Unknown UnknownAscusb_205 − − + − − + − − − Unknown Unknown Ascusb_232 − − − + − + − −− Unknown Unknown Ascusb_268 − − − − − + − − − Unknown UnknownAscusb_278 − − − − − + − + + Unknown Unknown Ascusb_329 − − − + − − − −− Unknown Unknown Ascusb_368 − − − − − + − − − Unknown UnknownAscusb_374 + − − + + + − − + Unknown Unknown Ascusb_411 − + − − − − − −− Unknown Unknown Ascusb_546 − + − − − + − − − Unknown UnknownAscusb_728 + − − + + + − − + Unknown Unknown Ascusb_765 − − − − − + −− + Unknown Unknown Ascusb_810 + − − − − + − − − Unknown UnknownAscusb_812 − − + − − − − − − Unknown Unknown Ascusb_817 − − + − + + −− + Unknown Unknown Ascusb_826 + − − + − + − − + Unknown UnknownAscusb_880 + − − + − + − + + Unknown Unknown Ascusb_913 + + − + − + − +− Unknown Unknown Ascusb_974 − + − − − + − − − Unknown UnknownAscusb_1069 − − − − − − − − + Unknown Unknown Ascusb_1074 − + + − − + −− − Unknown Unknown Ascusb_1295 + − − − + + − − + Unknown UnknownAscusb_1367 + + − − − + − + + Unknown Unknown Ascusb_1632 − − − − − + −− − Unknown Unknown Ascusb_1674 + − − + + − + − + Unknown UnknownAscusb_1763 + − − − − + − − − Unknown Unknown Ascusb_1780 − − − − + + −− + Unknown Unknown Ascusb_1786 + − − − − − − − − Unknown UnknownAscusb_1801 − − + − − + − − − Unknown Unknown Ascusb_1812 + − − − − − −− − Unknown Unknown Ascusb_1833 − + + + + + − − + Unknown UnknownAscusb_1850 − − − − + + − − + Unknown Unknown Ascusb_2090 + − − − − − −− + Unknown Unknown Ascusb_2124 + − − − − − − − − Unknown UnknownAscusb_2511 − + − + − + − − + Unknown Unknown Ascusb_2530 + − − − − + −− − Unknown Unknown Ascusb_2597 − + − − − + − − − Unknown UnknownAscusb_2624 − − − − − + − − − Unknown Unknown Ascusb_2667 + − − − − + −− + Unknown Unknown Ascusb_2836 − − − − + + − − + Unknown UnknownAscusb_3003 + − − + − − − − + Unknown Unknown Ascusb_3138 + − + − − +− + + Unknown Unknown Ascusb_3504 + − − − − + − − − Unknown UnknownAscusb_3881 − + − − − + − − − Unknown Unknown Ascusb_6589 − − + − − − −− − Unknown Unknown Ascusb_12103 + − − − − − − − + Unknown UnknownAscusb_14245 + − − − − + − − + Unknown Unknown Ascusb_20083 − − + − − −− − − Unknown Unknown Ascusb_20187 + − − − − + − − − Unknown UnknownAscusf_15 + + Unknown + + Unknown + + + + + Ascusf_22 − − Unknown − −Unknown − Unknown − + − Ascusf_23 + − Unknown − − Unknown − Unknown + +− Ascusf_24 − − Unknown − − Unknown − Unknown − + − Ascusf_25 + −Unknown − − Unknown − Unknown + − − Ascusf_38 − − Unknown − − Unknown −Unknown − + − Ascusf_45 + − Unknown − − Unknown − Unknown + + +Ascusf_77 + − Unknown + − Unknown − Unknown + + + Ascusf_94 + +Unknown + − Unknown − Unknown + + + Ascusf_108 + − Unknown − − Unknown −Unknown + − − Ascusf_206 − − Unknown − − Unknown − Unknown − + −Ascusf_208 − − Unknown − − Unknown − Unknown − + − Ascusf_307 + −Unknown − − Unknown − Unknown + + + Ascusf_334 + + Unknown + + Unknown −Unknown + + + Ascusf_353 + − Unknown + − Unknown − Unknown + − −Ascusf_1012 − − Unknown − − Unknown − Unknown − + −

TABLE 17 M2GSC and M2 Media Recipes M2GSC M2 Component Amount ComponentAmount Beef Extract 5 g NaHCO₃ 4 g Yeast Extract 1.25 g HCl-L-cysteine0.3 g NaHCO₃ 2 g (NH₄)₂SO₄ 0.10 g Cellobiose 1 g MgSO₄7H₂O 0.005 gStarch 1 g K₂HPO₄ 0.05 g Glucose 1 g KH₂PO₄ 0.05 g (NH₄)₂SO₄ (1M) 2.55mL DI H₂O Up to 1000 mL MgSO₄7H₂O (0.25M) 0.288 mL K₂HPO₄ (1M) 1 mLKH₂PO₄ (1M) 1.275 mL Clarified Rumen Fluid 50 mL HCl-L-cysteine 0.3 g DIH₂O Up to 500 mL

TABLE 18 Modified Wolfe's Media Recipes 250X Modified Wolfe's ModifiedWolfe's Vitamin Mix Mineral Solution Component g/200 mL Component g/LPyridoxine-HCl 0.5 MgSO₄ 7H₂O 140 p-Aminobenzoic 0.25 Nitrilotriaceticacid 10.96 Lipoic Acid 0.216 NaCl 50.06 Nicotinic Acid 0.252 MnSO₄ H₂O24.99 Riboflavin 0.013 CaCl₂ 5 Thiamine HCL 0.25 CoCl₂ 6H₂O 4.997Calcium - DL - 0.1 FeSO₂ 7H₂O 4.997 Pantothenate Biotin 0.044 ZnSO₂ 7H₂O5.003 Folic Acid 0.004 AlK(SO₄)₂ 12 H₂O 0.5 Vitamin B12 0.007 CuSO₄ 5H₂O0.499 H₃BO₃ 0.498 NaMoO₄ 2H₂O 0.503 DI H₂O 1 L

All media was prepared with anaerobic water (boiled DI H₂O for 15minutes then cooled to room temperature in a water bath while spargingwith N₂. All media was adjusted to a pH of 6.8 with 2M HCl. 10 mL ofmedia was then aliquoted into 15 mL hungate tubs, and the tubes werethen sparged with 80% N₂ 20% CO₂ for 3 minutes.

TABLE 19 RAMM Salts Media Recipe Component g/500 mL KH₂PO₄ 0.11 K₂HPO₄0.08 NH₄Cl 0.265 NaHCO₃ 0.6 DI H₂O 500 mL

After sterilization (autoclave) added: 2 mL of 250× modified Wolfe'svitamin mix, 10 mL of 50× modified Wolfe's mineral mix, 5 mL of 100 mMcysteine.

Example VIII Determination of Maximal Information Coefficient (MIC)Scores for Microbe Strains Relevant to Pounds of Milk ProducedExperimental Design and Materials and Methods

Objective: Determine rumen microbial community constituents that impactthe production of milk fat in dairy cows.

Animals: Eight lactating, ruminally cannulated, Holstein cows werehoused in individual tie-stalls for use in the experiment. Cows were fedtwice daily, milked twice a day, and had continuous access to freshwater. One cow (cow 1) was removed from the study after the firstdietary Milk Fat Depression due to complications arising from anabortion prior to the experiment.

Experimental Design and Treatment: The experiment used a crossoverdesign with 2 groups and 1 experimental period. The experimental periodlasted 38 days: 10 days for the covariate/wash-out period and 28 daysfor data collection and sampling. The data collection period consistedof 10 days of dietary Milk Fat Depression (MFD) and 18 days of recovery.After the first experimental period, all cows underwent a 10-day washout period prior to the beginning of period 2.

Dietary MFD was induced with a total mixed ration (TMR) low in fiber(29% NDF) with high starch degradability (70% degradable) and highpolyunsaturated fatty acid levels (PUFA, 3.7%). The Recovery phaseincluded two diets variable in starch degradability. Four cows wererandomly assigned to the recovery diet high in fiber (37% NDF), low inPUFA (2.6%), and high in starch degradability (70% degradable). Theremaining four cows were fed a recovery diet high in fiber (37% NDF),low in PUFA (2.6%), but low in starch degradability (35%).

During the 10-day covariate and 10-day wash out periods, cows were fedthe high fiber, low PUFA, and low starch degradability diet.

Samples and Measurements: Milk yield, dry matter intake, and feedefficiency were measured daily for each animal throughout the covariate,wash out, and sample collection periods. TMR samples were measured fornutrient composition. During the collection period, milk samples werecollected and analyzed every 3 days. Samples were analyzed for milkcomponent concentrations (milk fat, milk protein, lactose, milk ureanitrogen, somatic cell counts, and solids) and fatty acid compositions.

Rumen samples were collected and analyzed for microbial communitycomposition and activity every 3 days during the collection period. Therumen was intensively sampled 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and22 hours after feeding during day 0, day 7, and day 10 of the dietaryMFD. Similarly, the rumen was intensively sampled 0, 2, 4, 6, 8, 10, 12,14, 16, 18, 20, and 22 hours after feeding on day 16 and day 28 duringthe recovery period. Rumen contents were analyzed for pH, acetateconcentration, butyrate concentration, propionate concentration, isoacidconcentration, and long chain and CLA isomer concentrations.

Rumen Sample Preparation and Sequencing: After collection, rumen sampleswere centrifuged at 4,000 rpm in a swing bucket centrifuge for 20minutes at 4° C. The supernatant was decanted, and an aliquot of eachrumen content sample (1-2 mg) was added to a sterile 1.7 mL tubeprefilled with 0.1 mm glass beads. A second aliquot was collected andstored in an empty, sterile 1.7 mL tube for cell counting.

Rumen samples with glass beads (1^(st) aliquot) were homogenized withbead beating to lyse microorganisms. DNA and RNA was extracted andpurified from each sample and prepared for sequencing on an IlluminaMiseq. Samples were sequenced using paired-end chemistry, with 300 basepairs sequenced on each end of the library. Rumen samples in empty tubes(2^(nd) aliquot) were stained and put through a flow cytometer toquantify the number of cells of each microorganism type in each sample.

Sequencing Read Processing and Data Analysis: Sequencing reads werequality trimmed and processed to identify bacterial species present inthe rumen based on a marker gene. Count datasets and activity datasetswere integrated with the sequencing reads to determine the absolute cellnumbers of active microbial species within the rumen microbialcommunity. Production characteristics of the cow over time, includingpounds of milk produced, were linked to the distribution of activemicroorganisms within each sample over the course of the experimentusing mutual information. Maximal information coefficient (MIC) scoreswere calculated between pounds of milk fat produced and the absolutecell count of each active microorganism. Microorganisms were ranked byMIC score, and microorganisms with the highest MIC scores were selectedas the target species most relevant to pounds of milk produced.

Tests cases to determine the impact of count data, activity data, andcount and activity on the final output were run by omitting theappropriate datasets from the sequencing analysis. To assess the impactof using a linear correlation rather than the MIC on target selection,Pearson's coefficients were also calculated for pounds of milk fatproduced as compared to the relative abundance of all microorganisms andthe absolute cell count of active microorganisms.

Results and Discussion

Relative Abundances vs. Absolute Cell Counts

The top 15 target species were identified for the dataset that includedcell count data (absolute cell count, Table 21) and for the dataset thatdid not include cell count data (relative abundance, Table 20) based onMIC scores. Activity data was not used in this analysis in order toisolate the effect of cell count data on final target selection.Ultimately, the top 8 targets were the same between the two datasets. Ofthe remaining 7, 5 strains were present on both lists in varying order.Despite the differences in rank for these 5 strains, the calculated MICscore for each strain was the identical between the two lists. The twostrains present on the absolute cell count list but not the relativeabundance list, ascus_111 and ascus_288, were rank 91 and rank 16,respectively, on the relative abundance list. The two strains present onthe relative abundance list but not the absolute cell count list,ascus_102 and ascus_252, were rank 50 and rank 19, respectively, on theabsolute cell count list. These 4 strains did have different MIC scoreson each list, thus explaining their shift in rank and subsequent impacton the other strains in the list.

TABLE 20 Top 15 Target Strains using Relative Abundance with no ActivityFilter Target Strain MIC Nearest Taxonomy ascus_7 0.97384 d:Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756), o:Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g:Ruminococcus(0.0605) ascus_82 0.97173 d: Bacteria(1.0000), p:Firmicutes(0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714), f:Ruminococcaceae(0.1062), g: Saccharofermentans(0.0073) ascus_209 0.95251d: Bacteria(1.0000), p: TM7(0.9991), g:TM7_genera_incertae_sedis(0.8645) ascus_126 0.91477 d: Bacteria(1.0000),p: Firmicutes(0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714),f: Ruminococcaceae(0.1242), g: Saccharofermentans(0.0073) ascus_13660.89713 d: Bacteria(1.0000), p: TM7(0.9445), g:TM7_genera_incertae_sedis(0.0986) ascus_1780 0.89466 d:Bacteria(0.9401), p: Bacteroidetes(0.4304), c: Bacteroidia(0.0551), o:Bacteroidales(0.0198), f: Prevotellaceae(0.0067), g: Prevotella(0.0052)ascus_64 0.89453 d: Bacteria(1.0000), p: Firmicutes(0.9922), c:Clostridia(0.8823), o: Clostridiales(0.6267), f:Ruminococcaceae(0.2792), g: Ruminococcus(0.0605) ascus_299 0.88979 d:Bacteria(1.0000), p: TM7(0.9963), g: TM7_genera_incertae_sedis(0.5795)ascus_102 0.87095 d: Bacteria(1.0000), p: Firmicutes(0.9628), c:Clostridia(0.8317), o: Clostridiales(0.4636), f:Ruminococcaceae(0.2367), g: Saccharofermentans(0.0283) ascus_18010.87038 d: Bacteria(0.8663), p: Bacteroidetes(0.2483), c:Bacteroidia(0.0365), o: Bacteroidales(0.0179), f:Porphyromonadaceae(0.0059), g: Butyricimonas(0.0047) ascus_295 0.86724d: Bacteria(1.0000), p: SR1(0.9990), g:SR1_genera_incertae_sedis(0.9793) ascus_1139 0.8598 d: Bacteria(1.0000),p: TM7(0.9951), g: TM7_genera_incertae_sedis(0.4747) ascus_127 0.84082d: Bacteria(1.0000), p: TM7(0.9992), g:TM7_genera_incertae_sedis(0.8035) ascus_341 0.8348 d: Bacteria(1.0000),p: TM7(0.9992), g: TM7_genera_incertae_sedis(0.8035) ascus_252 0.82891d: Bacteria(1.0000), p: Firmicutes(0.9986), c: Clostridia(0.9022), o:Clostridiales(0.7491), f: Lachnospiraceae(0.3642), g:Lachnospiracea_incertae_sedis(0.0859)

TABLE 21 Top 15 Target Strains using Absolute cell count with noActivity Filter Target Strain MIC Nearest Taxonomy ascus_7 0.97384 d:Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756), o:Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g:Ruminococcus(0.0605) ascus_82 0.97173 d: Bacteria(1.0000), p:Firmicutes(0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714), f:Ruminococcaceae(0.1062), g: Saccharofermentans(0.0073) ascus_209 0.95251d: Bacteria(1.0000), p: TM7(0.9991), g:TM7_genera_incertae_sedis(0.8645) ascus_126 0.91701 d: Bacteria(1.0000),p: Firmicutes(0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714),f: Ruminococcaceae(0.1242), g: Saccharofermentans(0.0073) ascus_13660.89713 d: Bacteria(1.0000), p: TM7(0.9445), g:TM7_genera_incertae_sedis(0.0986) ascus_1780 0.89466 d:Bacteria(0.9401), p: Bacteroidetes(0.4304), c: Bacteroidia(0.0551), o:Bacteroidales(0.0198), f: Prevotellaceae(0.0067), g: Prevotella(0.0052)ascus_64 0.89453 d: Bacteria(1.0000), p: Firmicutes(0.9922), c:Clostridia(0.8823), o: Clostridiales(0.6267), f:Ruminococcaceae(0.2792), g: Ruminococcus(0.0605) ascus_299 0.88979 d:Bacteria(1.0000), p: TM7(0.9963), g: TM7_genera_incertae_sedis(0.5795)ascus_1801 0.87038 d: Bacteria(0.8663), p: Bacteroidetes(0.2483), c:Bacteroidia(0.0365), o: Bacteroidales(0.0179), f:Porphyromonadaceae(0.0059), g: Butyricimonas(0.0047) ascus_295 0.86724d: Bacteria(1.0000), p: SR1(0.9990), g:SR1_genera_incertae_sedis(0.9793) ascus_1139 0.8598 d: Bacteria(1.0000),p: TM7(0.9951), g: TM7_genera_incertae_sedis(0.4747) ascus_127 0.84082d: Bacteria(1.0000), p: TM7(0.9992), g:TM7_genera_incertae_sedis(0.8035) ascus_341 0.8348 d: Bacteria(1.0000),p: TM7(0.9992), g: TM7_genera_incertae_sedis(0.8035) ascus_111 0.83358d: Bacteria(1.0000), p: Firmicutes(0.7947), c: Clostridia(0.4637), o:Clostridiales(0.2335), f: Ruminococcaceae(0.1062), g:Papillibacter(0.0098) ascus_288 0.82833 d: Bacteria(0.7925), p:Bacteroidetes(0.2030), c: Bacteroidia(0.0327), o: Bacteroidales(0.0160),f: Porphyromonadaceae(0.0050), g: Butyricimonas(0.0042)

Integration of cell count data did not always affect the final MIC scoreassigned to each strain. This may be attributed to the fact thatalthough the microbial population did shift within the rumen daily andover the course of the 38-day experiment, it was always within 10⁷-10⁸cells per milliliter. Much larger shifts in population numbers wouldundoubtedly have a broader impact on final MIC scores.

Inactive Species vs. Active Species

In order to assess the impact of filtering strains based on activitydata, target species were identified from a dataset that leveragedrelative abundance with (Table 22) and without (Table 20) activity dataas well as a dataset that leveraged absolute cell counts with (Table 23)and without (Table 21) activity data.

For the relative abundance case, ascus_126, ascus_1366, ascus_1780,ascus_299, ascus_1139, ascus_127, ascus_341, and ascus_252 were deemedtarget strains prior to applying activity data. These eight strains (53%of the initial top 15 targets) fell below rank 15 after integratingactivity data. A similar trend was observed for the absolute cell countcase. Ascus_126, ascus_1366, ascus_1780, ascus_299, ascus_1139,ascus_127, and ascus_341 (46% of the initial top 15 targets) fell belowrank 15 after activity dataset integration.

The activity datasets had a much more severe effect on target rank andselection than the cell count datasets. When integrating these datasetstogether, if a sample is found to be inactive it is essentially changedto a “0” and not considered to be part of the analysis. Because of this,the distribution of points within a sample can become heavily altered orskewed after integration, which in turn greatly impacts the final MICscore and thus the rank order of target microorganisms.

TABLE 22 Top 15 Target Strains using Relative Abundance with ActivityFilter Target Strain MIC Nearest Taxonomy ascus_7 0.97384 d:Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756), o:Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g:Ruminococcus(0.0605) ascus_82 0.93391 d: Bacteria(1.0000), p:Firmicutes(0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714), f:Ruminococcaceae(0.1062), g: Saccharofermentans(0.0073) ascus_102 0.87095d: Bacteria(1.0000), p: Firmicutes(0.9628), c: Clostridia(0.8317), o:Clostridiales(0.4636), f: Ruminococcaceae(0.2367), g:Saccharofermentans(0.0283) ascus_209 0.84421 d: Bacteria(1.0000), p:TM7(0.9991), g: TM7_genera_incertae_sedis(0.8645) ascus_1801 0.82398 d:Bacteria(0.8663), p: Bacteroidetes(0.2483), c: Bacteroidia(0.0365), o:Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059), g:Butyricimonas(0.0047) ascus_372 0.81735 d: Bacteria(1.0000), p:Spirochaetes(0.9445), c: Spirochaetes(0.8623), o:Spirochaetales(0.5044), f: Spirochaetaceae(0.3217), g:Spirochaeta(0.0190) ascus_26 0.81081 d: Bacteria(1.0000), p:Firmicutes(0.9080), c: Clostridia(0.7704), o: Clostridiales(0.4230), f:Ruminococcaceae(0.1942), g: Clostridium_IV(0.0144) ascus_180 0.80702 d:Bacteria(1.0000), p: Spirochaetes(0.9445), c: Spirochaetes(0.8623), o:Spirochaetales(0.5044), f: Spirochaetaceae(0.3217), g:Spirochaeta(0.0237) ascus_32 0.7846 d: Bacteria(1.0000), p:Firmicutes(0.7036), c: Clostridia(0.4024), o: Clostridiales(0.1956), f:Ruminococcaceae(0.0883), g: Hydrogenoanaerobacterium(0.0144) ascus_2880.78229 d: Bacteria(0.7925), p: Bacteroidetes(0.2030), c:Bacteroidia(0.0327), o: Bacteroidales(0.0160), f:Porphyromonadaceae(0.0050), g: Butyricimonas(0.0042) ascus_64 0.77514 d:Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8823), o:Clostridiales(0.6267), f: Ruminococcaceae(0.2792), g:Ruminococcus(0.0605) ascus_295 0.76639 d: Bacteria(1.0000), p:SR1(0.9990), g: SR1_genera_incertae_sedis(0.9793) ascus_546 0.76114 d:Bacteria(1.0000), p: Firmicutes(0.6126), c: Clostridia(0.2851), o:Clostridiales(0.1324), f: Clostridiaceae_1(0.0208), g:Clostridium_sensu_stricto(0.0066) ascus_233 0.75779 d: Bacteria(1.0000),p: Firmicutes(0.9922), c: Clostridia(0.8756), o: Clostridiales(0.5860),f: Ruminococcaceae(0.3642), g: Ruminococcus(0.0478) ascus_651 0.74837 d:Bacteria(1.0000), p: Firmicutes(0.7947), c: Clostridia(0.4637), o:Clostridiales(0.2335), f: Ruminococcaceae(0.0883), g:Clostridium_IV(0.0069)

TABLE 23 Top 15 Target Strains using Absolute cell count with ActivityFilter Target Strain MIC Nearest Taxonomy ascus_7 0.97384 d:Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756), o:Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g:Ruminococcus(0.0605) ascus_82 0.93391 d: Bacteria(1.0000), p:Firmicutes(0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714), f:Ruminococcaceae(0.1062), g: Saccharofermentans(0.0073) ascus_209 0.84421d: Bacteria(1.0000), p: TM7(0.9991), g:TM7_genera_incertae_sedis(0.8645) ascus_1801 0.82398 d:Bacteria(0.8663), p: Bacteroidetes(0.2483), c: Bacteroidia(0.0365), o:Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059), g:Butyricimonas(0.0047) ascus_372 0.81735 d: Bacteria(1.0000), p:Spirochaetes(0.9445), c: Spirochaetes(0.8623), o:Spirochaetales(0.5044), f: Spirochaetaceae(0.3217), g:Spirochaeta(0.0190) ascus_26 0.81081 d: Bacteria(1.0000), p:Firmicutes(0.9080), c: Clostridia(0.7704), o: Clostridiales(0.4230), f:Ruminococcaceae(0.1942), g: Clostridium_IV(0.0144) ascus_102 0.81048 d:Bacteria(1.0000), p: Firmicutes(0.9628), c: Clostridia(0.8317), o:Clostridiales(0.4636), f: Ruminococcaceae(0.2367), g:Saccharofermentans(0.0283) ascus_111 0.79035 d: Bacteria(1.0000), p:Firmicutes(0.7947), c: Clostridia(0.4637), o: Clostridiales(0.2335), f:Ruminococcaceae(0.1062), g: Papillibacter(0.0098) ascus_288 0.78229 d:Bacteria(0.7925), p: Bacteroidetes(0.2030), c: Bacteroidia(0.0327), o:Bacteroidales(0.0160), f: Porphyromonadaceae(0.0050), g:Butyricimonas(0.0042) ascus_64 0.77514 d: Bacteria(1.0000), p:Firmicutes(0.9922), c: Clostridia(0.8823), o: Clostridiales(0.6267), f:Ruminococcaceae(0.2792), g: Ruminococcus(0.0605) ascus_295 0.76639 d:Bacteria(1.0000), p: SR1(0.9990), g: SR1_genera_incertae_sedis(0.9793)ascus_546 0.76114 d: Bacteria(1.0000), p: Firmicutes(0.6126), c:Clostridia(0.2851), o: Clostridiales(0.1324), f:Clostridiaceae_1(0.0208), g: Clostridium_sensu_stricto(0.0066) ascus_320.75068 d: Bacteria(1.0000), p: Firmicutes(0.7036), c:Clostridia(0.4024), o: Clostridiales(0.1956), f:Ruminococcaceae(0.0883), g: Hydrogenoanaerobacterium(0.0144) ascus_6510.74837 d: Bacteria(1.0000), p: Firmicutes(0.7947), c:Clostridia(0.4637), o: Clostridiales(0.2335), f:Ruminococcaceae(0.0883), g: Clostridium_IV(0.0069) ascus_233 0.74409 d:Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756), o:Clostridiales(0.5860), f: Ruminococcaceae(0.3642), g:Ruminococcus(0.0478)Relative Abundances and Inactive vs. Absolute Cell Counts and Active

Ultimately, the method defined here leverages both cell count data andactivity data to identify microorganisms highly linked to relevantmetadata characteristics. Within the top 15 targets selected using bothmethods (Table 23, Table 20), only 7 strains were found on both lists.Eight strains (53%) were unique to the absolute cell count and activitylist. The top 3 targets on both lists matched in both strain as well asin rank. However, two of the three did not have the same MIC score onboth lists, suggesting that they were influenced by activity datasetintegration but not enough to upset their rank order.

Linear Correlations vs. Nonparametric Approaches

Pearson's coefficients and MIC scores were calculated between pounds ofmilk fat produced and the absolute cell count of active microorganismswithin each sample (Table 24). Strains were ranked either by MIC (Table24a) or Pearson coefficient (Table 24b) to select target strains mostrelevant to milk fat production. Both MIC score and Pearson coefficientare reported in each case. Six strains were found on both lists, meaningnine (60%) unique strains were identified using the MIC approach. Therank order of strains between lists did not match—the top 3 targetstrains identified by each method were also unique.

Like Pearson coefficients, the MIC score is reported over a range of 0to 1, with 1 suggesting a very tight relationship between the twovariables. Here, the top 15 targets exhibited MIC scores ranging from0.97 to 0.74. The Pearson coefficients for the correlation test case,however, ranged from 0.53 to 0.45—substantially lower than the mutualinformation test case. This discrepancy may be due to the differencesinherent to each analysis method. While correlations are a linearestimate that measures the dispersion of points around a line, mutualinformation leverages probability distributions and measures thesimilarity between two distributions. Over the course of the experiment,the pounds of milk fat produced changed nonlinearly (FIG. 18). Thisparticular function may be better represented and approximated by mutualinformation than correlations. To investigate this, the top targetstrains identified using correlation and mutual information, Ascus_713(FIG. 19) and Ascus_7 (FIG. 20) respectively, were plotted to determinehow well each method predicted relationships between the strains andmilk fat. If two variables exhibit strong correlation, they arerepresented by a line with little to no dispersion of points whenplotted against each other. In FIG. 19, Ascus_713 correlates weakly withmilk fat, as indicated by the broad spread of points. Mutualinformation, again, measures how similar two distributions of pointsare. When Ascus_7 is plotted with milk fat (FIG. 20), it is apparentthat the two point distributions are very similar.

The Present Method in Entirety vs. Conventional Approaches

The conventional approach of analyzing microbial communities relies onthe use of relative abundance data with no incorporation of activityinformation, and ultimately ends with a simple correlation of microbialspecies to metadata (see, e.g., U.S. Pat. No. 9,206,680, which is hereinincorporated by reference in its entirety for all purposes). Here, wehave shown how the incorporation of each dataset incrementallyinfluences the final list of targets. When applied in its entirety, themethod described herein selected a completely different set of targetswhen compared to the conventional method (Table 24a and Table 24c).Ascus_3038, the top target strain selected using the conventionalapproach, was plotted against milk fat to visualize the strength of thecorrelation (FIG. 21). Like the previous example, Ascus_3038 alsoexhibited a weak correlation to milk fat.

Table 24: Top 15 Target Strains using Mutual Information or Correlations

TABLE 24a MIC using Absolute cell count with Activity Filter TargetPearson Strain MIC Coefficient Nearest Taxonomy ascus_7 0.973840.25282502 d: Bacteria(1.0000), p: Firmicutes(0.9922), c:Clostridia(0.8756), o: Clostridiales(0.5860), f:Ruminococcaceae(0.3217), g: Ruminococcus(0.0605) ascus_82 0.933910.42776647 d: Bacteria(1.0000), p: Firmicutes(0.8349), c:Clostridia(0.5251), o: Clostridiales(0.2714), f:Ruminococcaceae(0.1062), g: Saccharofermentans(0.0073) ascus_209 0.844210.3036308 d: Bacteria(1.0000), p: TM7(0.9991), g:TM7_genera_incertae_sedis(0.8645) ascus_1801 0.82398 0.5182261 d:Bacteria(0.8663), p: Bacteroidetes(0.2483), c: Bacteroidia(0.0365), o:Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059), g:Butyricimonas(0.0047) ascus_372 0.81735 0.34172258 d: Bacteria(1.0000),p: Spirochaetes(0.9445), c: Spirochaetes(0.8623), o:Spirochaetales(0.5044), f: Spirochaetaceae(0.3217), g:Spirochaeta(0.0190) ascus_26 0.81081 0.5300298 d: Bacteria(1.0000), p:Firmicutes(0.9080), c: Clostridia(0.7704), o: Clostridiales(0.4230), f:Ruminococcaceae(0.1942), g: Clostridium_IV(0.0144) ascus_102 0.810480.35456932 d: Bacteria(1.0000), p: Firmicutes(0.9628), c:Clostridia(0.8317), o: Clostridiales(0.4636), f:Ruminococcaceae(0.2367), g: Saccharofermentans(0.0283) ascus_111 0.790350.45881805 d: Bacteria(1.0000), p: Firmicutes(0.7947), c:Clostridia(0.4637), o: Clostridiales(0.2335), f:Ruminococcaceae(0.1062), g: Papillibacter(0.0098) ascus_288 0.782290.46522045 d: Bacteria(0.7925), p: Bacteroidetes(0.2030), c:Bacteroidia(0.0327), o: Bacteroidales(0.0160), f:Porphyromonadaceae(0.0050), g: Butyricimonas(0.0042) ascus_64 0.775140.45417055 d: Bacteria(1.0000), p: Firmicutes(0.9922), c:Clostridia(0.8823), o: Clostridiales(0.6267), f:Ruminococcaceae(0.2792), g: Ruminococcus(0.0605) ascus_295 0.766390.24972263 d: Bacteria(1.0000), p: SR1(0.9990), g:SR1_genera_incertae_sedis(0.9793) ascus_546 0.76114 0.23819838 d:Bacteria(1.0000), p: Firmicutes(0.6126), c: Clostridia(0.2851), o:Clostridiales(0.1324), f: Clostridiaceae_1(0.0208), g:Clostridium_sensu_stricto(0.0066) ascus_32 0.75068 0.5179697 d:Bacteria(1.0000), p: Firmicutes(0.7036), c: Clostridia(0.4024), o:Clostridiales(0.1956), f: Ruminococcaceae(0.0883), g:Hydrogenoanaerobacterium(0.0144) ascus_651 0.74837 0.27656645 d:Bacteria(1.0000), p: Firmicutes(0.7947), c: Clostridia(0.4637), o:Clostridiales(0.2335), f: Ruminococcaceae(0.0883), g:Clostridium_IV(0.0069) ascus_233 0.74409 0.36095098 d: Bacteria(1.0000),p: Firmicutes(0.9922), c: Clostridia(0.8756), o: Clostridiales(0.5860),f: Ruminococcaceae(0.3642), g: Ruminococcus(0.0478)

TABLE 24b Correlation using Absolute cell count with Activity FilterTarget Pearson Strain MIC Coefficient Nearest Taxonomy ascus_713 0.710660.5305876 d: Bacteria(1.0000), p: Firmicutes(0.8349), c:Clostridia(0.5251), o: Clostridiales(0.2714), f:Ruminococcaceae(0.1062), g: Saccharofermentans(0.0073) ascus_26 0.810810.5300298 d: Bacteria(1.0000), p: Firmicutes(0.9080), c:Clostridia(0.7704), o: Clostridiales(0.4230), f:Ruminococcaceae(0.1942), g: Clostridium_IV(0.0144) ascus_1801 0.823980.5182261 d: Bacteria(0.8663), p: Bacteroidetes(0.2483), c:Bacteroidia(0.0365), o: Bacteroidales(0.0179), f:Porphyromonadaceae(0.0059), g: Butyricimonas(0.0047) ascus_32 0.750680.5179697 d: Bacteria(1.0000), p: Firmicutes(0.7036), c:Clostridia(0.4024), o: Clostridiales(0.1956), f:Ruminococcaceae(0.0883), g: Hydrogenoanaerobacterium(0.0144) ascus_1190.6974 0.4968678 d: Bacteria(1.0000), p: Firmicutes(0.9922), c:Clostridia(0.8756), o: Clostridiales(0.5860), f:Ruminococcaceae(0.3217), g: Ruminococcus(0.0478) ascus_13899 0.645560.48739454 d: Bacteria(1.0000), p: Actinobacteria(0.1810), c:Actinobacteria(0.0365), o: Actinomycetales(0.0179), f:Propionibacteriaceae(0.0075), g: Microlunatus(0.0058) ascus_906 0.492560.48418677 d: Bacteria(1.0000), p: Firmicutes(0.8349), c:Clostridia(0.5251), o: Clostridiales(0.2714), f:Ruminococcaceae(0.1242), g: Papillibacter(0.0098) ascus_221 0.440060.47305903 d: Bacteria(1.0000), p: Bacteroidetes(0.9991), c:Bacteroidia(0.9088), o: Bacteroidales(0.7898), f:Prevotellaceae(0.3217), g: Prevotella(0.0986) ascus_1039 0.656290.46932846 d: Bacteria(1.0000), p: Firmicutes(0.7036), c:Clostridia(0.2851), o: Clostridiales(0.1324), f:Ruminococcaceae(0.0329), g: Clostridium_IV(0.0069) ascus_288 0.782290.46522045 d: Bacteria(0.7925), p: Bacteroidetes(0.2030), c:Bacteroidia(0.0327), o: Bacteroidales(0.0160), f:Porphyromonadaceae(0.0050), g: Butyricimonas(0.0042) ascus_589 0.408680.4651165 d: Bacteria(1.0000), p: Firmicutes(0.9981), c:Clostridia(0.9088), o: Clostridiales(0.7898), f:Lachnospiraceae(0.5986), g: Clostridium_XIVa(0.3698) ascus_41 0.672270.46499047 d: Bacteria(1.0000), p: Firmicutes(0.6126), c:Clostridia(0.3426), o: Clostridiales(0.1618), f:Ruminococcaceae(0.0703), g: Hydrogenoanaerobacterium(0.0098) ascus_1110.79035 0.45881805 d: Bacteria(1.0000), p: Firmicutes(0.7947), c:Clostridia(0.4637), o: Clostridiales(0.2335), f:Ruminococcaceae(0.1062), g: Papillibacter(0.0098) ascus_205 0.724410.45684373 d: Bacteria(1.0000), p: Firmicutes(0.6126), c:Clostridia(0.3426), o: Clostridiales(0.1618), f:Peptococcaceae_2(0.0449), g: Pelotomaculum(0.0069) ascus_64 0.775140.45417055 d: Bacteria(1.0000), p: Firmicutes(0.9922), c:Clostridia(0.8823), o: Clostridiales(0.6267), f:Ruminococcaceae(0.2792), g: Ruminococcus(0.0605)

TABLE 24c Correlation using Relative Abundance with no Activity FilterTarget Pearson Strain MIC Coefficient Nearest Taxonomy ascus_30380.56239 0.6007549 d: Bacteria(1.0000), p: Firmicutes(0.9945), c:Clostridia(0.8623), o: Clostridiales(0.5044), f:Lachnospiraceae(0.2367), g: Clostridium_XIVa(0.0350) ascus_1555 0.669650.59716415 d: Bacteria(1.0000), p: Firmicutes(0.7947), c:Clostridia(0.3426), o: Clostridiales(0.1618), f:Ruminococcaceae(0.0449), g: Clostridium_IV(0.0073) ascus_1039 0.685630.59292555 d: Bacteria(1.0000), p: Firmicutes(0.7036), c:Clostridia(0.2851), o: Clostridiales(0.1324), f:Ruminococcaceae(0.0329), g: Clostridium_IV(0.0069) ascus_1424 0.555090.57589555 d: Bacteria(1.0000), p: Firmicutes(0.8897), c:Clostridia(0.7091), o: Clostridiales(0.3851), f:Ruminococcaceae(0.1422), g: Papillibacter(0.0144) ascus_378 0.775190.5671971 d: Bacteria(1.0000), p: Firmicutes(0.8349), c:Clostridia(0.5251), o: Clostridiales(0.2714), f:Ruminococcaceae(0.1062), g: Saccharofermentans(0.0073) ascus_407 0.697830.56279755 d: Bacteria(1.0000), p: Firmicutes(0.7036), c:Clostridia(0.3426), o: Clostridiales(0.1618), f:Clostridiaceae_1(0.0329), g: Clostridium_sensu_stricto(0.0069)ascus_1584 0.5193 0.5619939 d: Bacteria(1.0000), p: Firmicutes(0.9945),c: Clostridia(0.8756), o: Clostridiales(0.5860), f:Lachnospiraceae(0.3217), g: Coprococcus(0.0605) ascus_760 0.613630.55807924 d: Bacteria(1.0000), p: Firmicutes(0.6126), c:Clostridia(0.2851), o: Clostridiales(0.1324), f:Clostridiaceae_1(0.0208), g: Clostridium_sensu_stricto(0.0066)ascus_1184 0.70593 0.5578006 d: Bacteria(1.0000), p:“Bacteroidetes”(0.9992), c: “Bacteroidia”(0.8690), o:“Bacteroidales”(0.5452), f: Bacteroidaceae(0.1062), g:Bacteroides(0.0237) ascus_7394 0.6269 0.5557023 d: Bacteria(1.0000), p:Firmicutes(0.9939), c: Clostridia(0.7704), o: Clostridiales(0.4230), f:Lachnospiraceae(0.1422), g: Clostridium_XIVa(0.0350) ascus_1360 0.573430.5535785 d: Bacteria(1.0000), p: Firmicutes(0.9992), c:Clostridia(0.9351), o: Clostridiales(0.8605), f:Lachnospiraceae(0.7052), g: Clostridium_XIVa(0.2649) ascus_3175 0.535650.54864305 d: Bacteria(1.0000), p: “Bacteroidetes”(0.9991), c:“Bacteroidia”(0.8955), o: “Bacteroidales”(0.7083), f:“Prevotellaceae”(0.1942), g: Prevotella(0.0605) ascus_2581 0.683610.5454486 d: Bacteria(1.0000), p: “Spirochaetes”(0.9445), c:Spirochaetes(0.8623), o: Spirochaetales(0.5044), f:Spirochaetaceae(0.3217), g: Spirochaeta(0.0237) ascus_531 0.713150.5400517 d: Bacteria(1.0000), p: Firmicutes(0.6126), c:Clostridia(0.2851), o: Clostridiales(0.1324), f:Clostridiaceae_1(0.0208), g: Clostridium_sensu_stricto(0.0066)ascus_1858 0.65165 0.5393882 d: Bacteria(1.0000), p:“Spirochaetes”(0.9263), c: Spirochaetes(0.8317), o:Spirochaetales(0.4636), f: Spirochaetaceae(0.2792), g:Spirochaeta(0.0237)

Numbered Embodiments of the Disclosure

Subject matter contemplated by the present disclosure is set out in thefollowing numbered embodiments:

-   -   1. A shelf-stable ruminant supplement capable of increasing milk        production or improving milk compositional characteristics in a        ruminant, comprising:        -   a) a purified population of Pichia fungi comprising a fungi            with an ITS nucleic acid sequence that is at least about 97%            identical to SEQ ID NO: 32; and        -   b) a shelf-stable carrier suitable for ruminant            administration,        -   wherein the purified population of Pichia fungi of a) is            present in the supplement in an amount effective to increase            milk production or improve milk compositional            characteristics in a ruminant administered the supplement,            as compared to a ruminant not administered the supplement.    -   2. The shelf-stable ruminant supplement according to claim 1,        wherein the purified population of Pichia fungi comprises a        fungi with an ITS nucleic acid sequence that is at least about        99% identical to SEQ ID NO: 32.    -   3. The shelf-stable ruminant supplement according to claim 1,        wherein the purified population of Pichia fungi comprises a        fungi with an ITS nucleic acid sequence comprising SEQ ID NO:        32.    -   4. The shelf-stable ruminant supplement according to claim 1,        wherein the purified population of Pichia fungi comprises a        fungi as deposited at NRRL Y-67249.    -   5. The shelf-stable ruminant supplement according to claim 1,        further comprising:        -   i. a purified population of bacteria that comprises a            bacteria with a 16S nucleic acid sequence that is at least            about 97% identical to a nucleic acid sequence selected from            the group consisting of: SEQ ID NOs: 1-30 and 2045-2103,            and/or        -   ii. a purified population of fungi that comprises a fungi            with an ITS nucleic acid sequence that is at least about 97%            identical to a nucleic acid sequence selected from the group            consisting of: SEQ ID NOs: 31, 33-60 and 2104-2107.    -   6. The shelf-stable ruminant supplement according to claim 5,        wherein the purified population of bacteria comprises a bacteria        with a 16S nucleic acid sequence that is at least about 99%        identical to a nucleic acid sequence selected from the group        consisting of: SEQ ID NOs: 1-30 and 2045-2103.    -   7. The shelf-stable ruminant supplement according to claim 5,        wherein the purified population of fungi comprises a fungi with        an ITS nucleic acid sequence that is at least about 99%        identical to a nucleic acid sequence selected from the group        consisting of: SEQ ID NOs: 31, 33-60 and 2104-2107.    -   8. The shelf-stable ruminant supplement according to claim 5,        wherein the purified population of bacteria comprises a bacteria        with a 16S nucleic acid sequence selected from the group        consisting of: SEQ ID NOs: 1-30 and 2045-2103.    -   9. The shelf-stable ruminant supplement according to claim 5,        wherein the purified population of fungi comprises a fungi with        an ITS nucleic acid sequence selected from the group consisting        of: SEQ ID NOs: 31, 33-60 and 2104-2107.    -   10. The shelf-stable ruminant supplement according to claim 5,        wherein the purified population of bacteria comprises a bacteria        with a 16S nucleic acid sequence that is at least about 97%        identical to SEQ ID NO: 28.    -   11. The shelf-stable ruminant supplement according to claim 5,        wherein the purified population of bacteria comprises a bacteria        with a 16S nucleic acid sequence that is at least about 99%        identical to SEQ ID NO: 28.    -   12. The shelf-stable ruminant supplement according to claim 5,        wherein the purified population of bacteria comprises a bacteria        with a 16S nucleic acid sequence comprising SEQ ID NO: 28.    -   13. The shelf-stable ruminant supplement according to claim 5,        wherein the purified population of bacteria comprises a bacteria        as deposited at NRRL B-67248.    -   14. The shelf-stable ruminant supplement according to claim 5,        wherein both a purified population of bacteria i) and a purified        population of fungi ii) are present in the supplement.    -   15. The shelf-stable ruminant supplement according to claim 1,        formulated for administration to a cow.    -   16. The shelf-stable ruminant supplement according to claim 1,        wherein the supplement is stable under ambient conditions for at        least one week.    -   17. The shelf-stable ruminant supplement according to claim 1,        formulated as an: encapsulation, tablet, capsule, pill, feed        additive, food ingredient, food additive, food preparation, food        supplement, consumable solution, consumable spray additive,        consumable solid, consumable gel, injection, suppository, bolus,        drench, or combinations thereof.    -   18. The shelf-stable ruminant supplement according to claim 1,        wherein the purified population of Pichia fungi is present in        the ruminant supplement at a concentration of at least 10²        cells.    -   19. The shelf-stable ruminant supplement according to claim 1,        wherein the ruminant administered the supplement exhibits an        increase in milk production that leads to a measured increase in        milk yield.    -   20. The shelf-stable ruminant supplement according to claim 1,        wherein the ruminant administered the supplement exhibits an        increase in milk production and improved milk compositional        characteristics that leads to a measured increase in        energy-corrected milk.    -   21. The shelf-stable ruminant supplement according to claim 1,        wherein the ruminant administered the supplement exhibits an        improved milk compositional characteristic selected from the        group consisting of: an increase in milk fat(s), an increase in        milk protein(s), an increase of carbohydrates in milk, an        increase of vitamins in milk, an increase of minerals in milk,        or combinations thereof.    -   22. The shelf-stable ruminant supplement according to claim 1,        wherein the ruminant administered the supplement exhibits at        least a 1% increase in the average production of: milk fat(s),        milk protein(s), energy-corrected milk, or combinations thereof.    -   23. The shelf-stable ruminant supplement according to claim 1,        wherein the ruminant administered the supplement exhibits at        least a 10% increase in the average production of: milk fat(s),        milk protein(s), energy-corrected milk, or combinations thereof.    -   24. The shelf-stable ruminant supplement according to claim 1,        wherein the ruminant administered the supplement exhibits at        least a 20% increase in the average production of: milk fat(s),        milk protein(s), energy-corrected milk, or combinations thereof.    -   25. A composition suitable for administration to a ruminant and        capable of increasing milk production or improving milk        compositional characteristics in a ruminant, comprising:        -   a) a purified population of fungi as deposited at NRRL            Y-67249; and        -   b) a carrier suitable for ruminant administration,        -   wherein the purified population of fungi of a) is present in            the composition in an amount effective to increase milk            production or improve milk compositional characteristics in            a ruminant administered the composition, as compared to a            ruminant not administered the composition.    -   26. A composition suitable for administration to a ruminant and        capable of increasing milk production or improving milk        compositional characteristics in a ruminant, comprising:        -   a) a purified population of fungi as deposited at NRRL            Y-67249;        -   b) a purified population of bacteria as deposited at NRRL            B-67248; and        -   c) a carrier suitable for ruminant administration,        -   wherein the purified population of fungi of a) and purified            population of bacteria of b) are present in the composition            in an amount effective to increase milk production or            improve milk compositional characteristics in a ruminant            administered the composition, as compared to a ruminant not            administered the composition.

The aforementioned compositions have markedly different characteristicsand/or properties not possessed by any individual bacteria or fungi asthey naturally exist in the rumen. The markedly differentcharacteristics and/or properties possessed by the aforementionedcompositions can be structural, functional, or both. For example, thecompositions possess the markedly different functional property of beingable to increase milk production or improve milk compositionalcharacteristics, when administered to a ruminant, as taught herein.Furthermore, the compositions possess the markedly different functionalproperty of being shelf-stable.

Numbered Embodiments of the Disclosure

Subject matter contemplated by the present disclosure is set out in thefollowing numbered embodiments:

-   -   1. A composition capable of modulating the rumen microbiome of a        ruminant, comprising:        -   a) a purified population of Pichia fungi comprising a fungi            with an ITS nucleic acid sequence that is at least about 97%            identical to SEQ ID NO: 32; and        -   b) a carrier suitable for ruminant administration,    -   wherein the purified population of Pichia fungi of a) is present        in the composition in an amount effective to cause a shift in        the microbiome of the rumen of a ruminant administered the        composition.    -   2. The composition according to claim 1, wherein a population of        microbes present in the ruminant's rumen before administration        of the composition increase in abundance after administration of        the composition.    -   3. The composition according to claim 1, wherein a population of        microbes present in the ruminant's rumen before administration        of the composition decrease in abundance after administration of        the composition.    -   4. The composition according to claim 1, wherein a first        population of microbes present in the ruminant's rumen before        administration of the composition increase in abundance after        administration of the composition and wherein a second        population of microbes present in the ruminant's rumen before        administration of the composition decrease in abundance after        administration of the composition.    -   5. The composition according to claim 1, wherein the rumen        microbiome of the ruminant administered the composition is        shifted to include an increased presence of fiber-degrading        genera, volatile fatty acid-producing genera, structural        carbohydrate-digesting genera, or combinations thereof.    -   6. The composition according to claim 1, wherein the rumen        microbiome of the ruminant administered the composition is        shifted according to the disclosure and data presented in        Example 6 and Table 13 or Table 14.    -   7. A method for modulating the rumen microbiome of a ruminant,        comprising administering to a ruminant an effective amount of a        composition comprising:        -   a) a purified microbial population, said purified microbial            population comprising:            -   i. a purified population of bacteria that comprises a                bacteria with a 16S nucleic acid sequence that is at                least about 97% identical to a nucleic acid sequence                selected from the group consisting of: SEQ ID NOs: 1-30                and 2045-2103, and/or            -   ii. a purified population of fungi that comprises a                fungi with an ITS nucleic acid sequence that is at least                about 97% identical to a nucleic acid sequence selected                from the group consisting of: SEQ ID NOs: 31-60 and                2104-2107; and        -   b) a carrier suitable for ruminant administration,        -   wherein the ruminant administered the effective amount of            the composition exhibits a shift in the microbiome of the            rumen.    -   8. The method according to claim 7, wherein a population of        microbes present in the ruminant's rumen before administration        of the composition increase in abundance after administration of        the composition.    -   9. The method according to claim 7, wherein a population of        microbes present in the ruminant's rumen before administration        of the composition decrease in abundance after administration of        the composition.    -   10. The method according to claim 7, wherein a first population        of microbes present in the ruminant's rumen before        administration of the composition increase in abundance after        administration of the composition and wherein a second        population of microbes present in the ruminant's rumen before        administration of the composition decrease in abundance after        administration of the composition.    -   11. The method according to claim 7, wherein the rumen        microbiome of the ruminant administered the composition is        shifted to include an increased presence of fiber-degrading        genera, volatile fatty acid-producing genera, structural        carbohydrate-digesting genera, or combinations thereof.    -   12. The method according to claim 7, wherein the rumen        microbiome of the ruminant administered the composition is        shifted according to the disclosure and data presented in        Example 6 and Table 13 or Table 14.

The aforementioned compositions have markedly different characteristicsand/or properties not possessed by any individual bacteria or fungi asthey naturally exist in the rumen. The markedly differentcharacteristics and/or properties possessed by the aforementionedcompositions can be structural, functional, or both. For example, thecompositions possess the markedly different functional property of beingable to modulate the rumen microbiome, when administered to a ruminant,as taught herein.

Numbered Embodiments of the Disclosure

Subject matter contemplated by the present disclosure is set out in thefollowing numbered embodiments:

-   -   1. A method for increasing milk production or improving milk        compositional characteristics in a ruminant, comprising:        -   a) administering to a ruminant an effective amount of a            shelf-stable ruminant supplement comprising:            -   i. a purified microbial population that comprises a                bacteria with a 16S nucleic acid sequence, and/or a                fungi with an ITS nucleic acid sequence, which is at                least about 97% identical to a nucleic acid sequence                selected from the group consisting of: SEQ ID NOs: 1-60                and 2045-2107, said bacteria having a MIC score of at                least about 0.4 and said fungi having a MIC score of at                least about 0.2; and            -   ii. a shelf-stable carrier suitable for ruminant                administration,        -   wherein at least one of the bacteria or fungi are capable of            converting a carbon source into a volatile fatty acid            selected from the group consisting of: acetate, butyrate,            propionate, or combinations thereof; and        -   wherein at least one of the bacteria or fungi are capable of            degrading a soluble or insoluble carbon source; and        -   wherein the ruminant administered the effective amount of            the shelf-stable ruminant supplement exhibits an increase in            milk production or improved milk compositional            characteristics, as compared to a ruminant not administered            the ruminant supplement.    -   2. The method according to claim 1, wherein the ruminant is a        cow.    -   3. The method according to claim 1, wherein the ruminant        supplement is stable under ambient conditions for at least one        week.    -   4. The method according to claim 1, wherein the ruminant        supplement is formulated as an: encapsulation, tablet, capsule,        pill, feed additive, food ingredient, food additive, food        preparation, food supplement, consumable solution, consumable        spray additive, consumable solid, consumable gel, injection,        suppository, bolus, drench, or combinations thereof.    -   5. The method according to claim 1, wherein the ruminant        supplement is encapsulated in a polymer or carbohydrate.    -   6. The method according to claim 1, wherein administering        comprises: feeding the ruminant supplement to a ruminant.    -   7. The method according to claim 1, wherein administering        comprises: injecting the ruminant supplement into a ruminant.    -   8. The method according to claim 1, wherein the purified        microbial population is present in the ruminant supplement at a        concentration of at least 10² cells.    -   9. The method according to claim 1, wherein the purified        microbial population comprises a bacteria with a 16S nucleic        acid sequence that is at least about 97% identical to a nucleic        acid sequence selected from the group consisting of: SEQ ID NOs:        1-30 and 2045-2103.    -   10. The method according to claim 1, wherein the purified        microbial population comprises a fungi with an ITS nucleic acid        sequence that is at least about 97% identical to a nucleic acid        sequence selected from the group consisting of: SEQ ID NOs:        31-60 and 2104-2107.    -   11. The method according to claim 1, wherein the purified        microbial population comprises a bacteria with a 16S nucleic        acid sequence that is at least about 99% identical to a nucleic        acid sequence selected from the group consisting of: SEQ ID NOs:        1-30 and 2045-2103.    -   12. The method according to claim 1, wherein the purified        microbial population comprises a fungi with an ITS nucleic acid        sequence that is at least about 99% identical to a nucleic acid        sequence selected from the group consisting of: SEQ ID NOs:        31-60 and 2104-2107.    -   13. The method according to claim 1, wherein the purified        microbial population comprises a bacteria with a 16S nucleic        acid sequence selected from the group consisting of: SEQ ID NOs:        1-30 and 2045-2103.    -   14. The method according to claim 1, wherein the purified        microbial population comprises a fungi with an ITS nucleic acid        sequence selected from the group consisting of: SEQ ID NOs:        31-60 and 2104-2107.    -   15. The method according to claim 1, wherein the purified        microbial population comprises a bacteria with a 16S nucleic        acid sequence and a fungi with an ITS nucleic acid sequence that        is at least about 97% identical to a nucleic acid sequence        selected from the group consisting of: SEQ ID NOs: 1-60 and        2045-2107.    -   16. The method according to claim 1, wherein the purified        microbial population comprises a bacteria with a 16S nucleic        acid sequence that is at least about 97% identical to SEQ ID NO:        28.    -   17. The method according to claim 1, wherein the purified        microbial population comprises a fungi with an ITS nucleic acid        sequence that is at least about 97% identical to SEQ ID NO: 32.    -   18. The method according to claim 1, wherein the purified        microbial population comprises a Pichia fungi as deposited at        NRRL Y-67249.    -   19. The method according to claim 1, wherein the purified        microbial population only contains organisms that are members of        a group selected from:        -   Intestinimonas, Anaerolinea, Pseudobutyrivibrio, Olsenella,            Eubacterium, Catenisphaera, Faecalibacterium, Solobacterium,            Blautia, Ralsonia, Coprococcus, Casaltella, Anaeroplasma,            Acholeplasma, Aminiphilus, Mitsuokella, Alistipes, Sharpea,            Oscillibacter, Neocallimastix, Odoribacter, Pichia,            Tannerella, Candida, Hydrogenoanaerobacterium, Orpinomyces,            Succinivibrio, Sugiyamaella, Ruminobacter, Lachnospira,            Caecomyces, Sinimarinibacterium, Tremella,            Hydrogenoanaerobacterium, Turicibacter, Clostridium_XIVa,            Anaerolinea, Saccharofermentans, Butyricicoccus, Olsenella,            Papillibacter, Clostridium XIa, Pelotomaculum,            Erysipelotrichaceae incertae sedis,            Lachnospiracea_incertae_sedis, Solobacterium, Anaeroplasma,            Ralstonia, Clostridium_sensu_stricto, Eubacterium,            Rikenella, Lachnobacterium, Tannerella, Acholeplasma,            Howardella, Selenomonas, Butyricimonas, Sharpea,            Succinivibrio, Ruminobacter, Candida, Syntrophococcus,            Pseudobtqrivibrio, Orpinomyces, Cyllamyces,            Saccharomycetales, Phyllosticta, Ascomycota, and Piromyces.    -   20. The method according to claim 1, wherein the ruminant        administered the effective amount of the ruminant supplement        exhibits an increase in milk production that leads to a measured        increase in milk yield.    -   21. The method according to claim 1, wherein the ruminant        administered the effective amount of the ruminant supplement        exhibits an increase in milk production and improved milk        compositional characteristics that leads to a measured increase        in energy-corrected milk.    -   22. The method according to claim 1, wherein the ruminant        administered the effective amount of the ruminant supplement        exhibits an improved milk compositional characteristic selected        from the group consisting of: an increase in milk fat(s), an        increase in milk protein(s), an increase of carbohydrates in        milk, an increase of vitamins in milk, an increase of minerals        in milk, or combinations thereof.    -   23. The method according to claim 1, wherein the ruminant        administered the effective amount of the ruminant supplement        exhibits at least a 1% increase in the average production of:        milk fat(s), milk protein(s), energy-corrected milk, or        combinations thereof.    -   24. The method according to claim 1, wherein the ruminant        administered the effective amount of the ruminant supplement        exhibits at least a 10% increase in the average production of:        milk fat(s), milk protein(s), energy-corrected milk, or        combinations thereof.    -   25. The method according to claim 1, wherein the ruminant        administered the effective amount of the ruminant supplement        exhibits at least a 20% increase in the average production of:        milk fat(s), milk protein(s), energy-corrected milk, or        combinations thereof.    -   26. The method according to claim 1, wherein the ruminant        administered the effective amount of the ruminant supplement,        further exhibits:        -   at least one improved phenotypic trait, selected from the            group consisting of: an improved efficiency in feed            utilization, improved digestibility, an increase in            polysaccharide and lignin degradation, an increase in fatty            acid concentration in the rumen, pH balance in the rumen, a            reduction in methane emissions, a reduction in manure            production, improved dry matter intake, an improved            efficiency of nitrogen utilization, or combinations thereof.    -   27. The method according to claim 1, wherein the ruminant        administered the effective amount of the ruminant supplement,        further exhibits: a shift in the microbiome of the rumen.    -   28. The method according to claim 1, wherein the ruminant        administered the effective amount of the ruminant supplement,        further exhibits: a shift in the microbiome of the rumen,        -   wherein a population of microbes present in the rumen before            administration of the ruminant supplement increase in            abundance after administration of the ruminant supplement.    -   29. The method according to claim 1, wherein the ruminant        administered the effective amount of the ruminant supplement,        further exhibits: a shift in the microbiome of the rumen,        -   wherein a population of microbes present in the rumen before            administration of the ruminant supplement decrease in            abundance after administration of the ruminant supplement.    -   30. The method according to claim 1, wherein the ruminant        administered the effective amount of the ruminant supplement,        further exhibits: a shift in the microbiome of the rumen,        -   wherein a first population of microbes present in the rumen            before administration of the ruminant supplement increase in            abundance after administration of the ruminant supplement,            and        -   wherein a second population of microbes present in the rumen            before administration of the ruminant supplement decrease in            abundance after administration of the ruminant supplement.

The aforementioned compositions, utilized in the described methods, havemarkedly different characteristics and/or properties not possessed byany individual bacteria or fungi as they naturally exist in the rumen.The markedly different characteristics and/or properties possessed bythe aforementioned compositions, utilized in the described methods, canbe structural, functional, or both. For example, the compositions,utilized in the described methods, possess the markedly differentfunctional property of being able to increase milk production or improvemilk compositional characteristics, when administered to a ruminant, astaught herein. Furthermore, the compositions, utilized in the describedmethods, possess the markedly different functional property of beingshelf-stable.

In aspects, the aforementioned microbial species—that is, a purifiedmicrobial population that comprises a bacteria with a 16S nucleic acidsequence, and/or a fungi with an ITS nucleic acid sequence, which is atleast about 97% identical to a nucleic acid sequence selected from thegroup consisting of: SEQ ID NOs: 1-60 and 2045-2107—are members of aMarkush group, as the present disclosure illustrates that the membersbelong to a class of microbes characterized by various physical andfunctional attributes, which can include any of the following: a) theability to convert a carbon source into a volatile fatty acid such asacetate, butyrate, propionate, or combinations thereof; b) the abilityto degrade a soluble or insoluble carbon source; c) the ability toimpart an increase in milk production or improved milk compositionalcharacteristics to a ruminant administered the microbe; d) the abilityto modulate the microbiome of the rumen of a ruminant administered themicrobe; e) the ability to be formulated into a shelf-stablecomposition; and/or f) possessing a MIC score of at least about 0.4 if abacteria and possessing a MIC score of at least about 0.2 if a fungi.Thus, the members of the Markush group possess at least one property incommon, which can be responsible for their function in the claimedrelationship.

TABLE 25 Budapest Treaty Deposits of the Disclosure Depository AccessionNumber Date of Deposit NRRL NRRL Y-67249 Apr. 27, 2016 NRRL NRRL B-67248Apr. 27, 2016 NRRL NRRL B-67347 Dec. 15, 2016 NRRL NRRL B-67348 Dec. 15,2016 NRRL NRRL B-67349 Dec. 15, 2016 Bigelow PATENT201612001 Dec. 12,2016 Bigelow PATENT201612002 Dec. 12, 2016 Bigelow PATENT201612003 Dec.12, 2016 Bigelow PATENT201612004 Dec. 12, 2016 Bigelow PATENT201612005Dec. 12, 2016 Bigelow PATENT201612006 Dec. 12, 2016 BigelowPATENT201612007 Dec. 15, 2016 Bigelow PATENT201612008 Dec. 15, 2016Bigelow PATENT201612009 Dec. 15, 2016 Bigelow PATENT201612010 Dec. 15,2016 Bigelow PATENT201612011 Dec. 15, 2016 Bigelow PATENT201612012 Dec.15, 2016 Bigelow PATENT201612013 Dec. 19, 2016 Bigelow PATENT201612014Dec. 28, 2016

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as, an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

What is claimed is:
 1. A method for increasing milk production orimproving milk compositional characteristics in a ruminant, comprising:a) administering to a ruminant an effective amount of a shelf-stableruminant supplement comprising: i. a purified microbial population thatcomprises a bacteria with a 16S nucleic acid sequence, and/or a fungiwith an ITS nucleic acid sequence, which is at least about 97% identicalto a nucleic acid sequence selected from the group consisting of: SEQ IDNOs: 1-60 and 2045-2107, said bacteria having a MIC score of at leastabout 0.4 and said fungi having a MIC score of at least about 0.2; andii. a shelf-stable carrier suitable for ruminant administration, whereinat least one of the bacteria or fungi are capable of converting a carbonsource into a volatile fatty acid selected from the group consisting of:acetate, butyrate, propionate, or combinations thereof; and wherein atleast one of the bacteria or fungi are capable of degrading a soluble orinsoluble carbon source; and wherein the ruminant administered theeffective amount of the shelf-stable ruminant supplement exhibits anincrease in milk production or improved milk compositionalcharacteristics, as compared to a ruminant not administered the ruminantsupplement.
 2. The method according to claim 1, wherein the ruminant isa cow.
 3. The method according to claim 1, wherein the ruminantsupplement is stable under ambient conditions for at least one week. 4.The method according to claim 1, wherein the ruminant supplement isformulated as an: encapsulation, tablet, capsule, pill, feed additive,food ingredient, food additive, food preparation, food supplement,consumable solution, consumable spray additive, consumable solid,consumable gel, injection, suppository, bolus, drench, or combinationsthereof.
 5. The method according to claim 1, wherein the ruminantsupplement is encapsulated in a polymer or carbohydrate.
 6. The methodaccording to claim 1, wherein administering comprises: feeding theruminant supplement to a ruminant.
 7. The method according to claim 1,wherein administering comprises: injecting the ruminant supplement intoa ruminant.
 8. The method according to claim 1, wherein the purifiedmicrobial population is present in the ruminant supplement at aconcentration of at least 10² cells.
 9. The method according to claim 1,wherein the purified microbial population comprises a bacteria with a16S nucleic acid sequence that is at least about 97% identical to anucleic acid sequence selected from the group consisting of: SEQ ID NOs:1-30 and 2045-2103.
 10. The method according to claim 1, wherein thepurified microbial population comprises a fungi with an ITS nucleic acidsequence that is at least about 97% identical to a nucleic acid sequenceselected from the group consisting of: SEQ ID NOs: 31-60 and 2104-2107.11. The method according to claim 1, wherein the purified microbialpopulation comprises a bacteria with a 16S nucleic acid sequence that isat least about 99% identical to a nucleic acid sequence selected fromthe group consisting of: SEQ ID NOs: 1-30 and 2045-2103.
 12. The methodaccording to claim 1, wherein the purified microbial populationcomprises a fungi with an ITS nucleic acid sequence that is at leastabout 99% identical to a nucleic acid sequence selected from the groupconsisting of: SEQ ID NOs: 31-60 and 2104-2107.
 13. The method accordingto claim 1, wherein the purified microbial population comprises abacteria with a 16S nucleic acid sequence selected from the groupconsisting of: SEQ ID NOs: 1-30 and 2045-2103.
 14. The method accordingto claim 1, wherein the purified microbial population comprises a fungiwith an ITS nucleic acid sequence selected from the group consisting of:SEQ ID NOs: 31-60 and 2104-2107.
 15. The method according to claim 1,wherein the purified microbial population comprises a bacteria with a16S nucleic acid sequence and a fungi with an ITS nucleic acid sequencethat is at least about 97% identical to a nucleic acid sequence selectedfrom the group consisting of: SEQ ID NOs: 1-60 and 2045-2107.
 16. Themethod according to claim 1, wherein the purified microbial populationcomprises a bacteria with a 16S nucleic acid sequence that is at leastabout 97% identical to SEQ ID NO:
 28. 17. The method according to claim1, wherein the purified microbial population comprises a fungi with anITS nucleic acid sequence that is at least about 97% identical to SEQ IDNO:
 32. 18. The method according to claim 1, wherein the purifiedmicrobial population comprises a Pichia fungi as deposited at NRRLY-67249.
 19. The method according to claim 1, wherein the purifiedmicrobial population only contains organisms that are members of a groupselected from: Intestinimonas, Anaerolinea, Pseudobutyrivibrio,Olsenella, Eubacterium, Catenisphaera, Faecalibacterium, Solobacterium,Blautia, Ralsonia, Coprococcus, Casaltella, Anaeroplasma, Acholeplasma,Aminiphilus, Mitsuokella, Alistipes, Sharpea, Oscillibacter,Neocallimastix, Odoribacter, Pichia, Tannerella, Candida,Hydrogenoanaerobacterium, Orpinomyces, Succinivibrio, Sugiyamaella,Ruminobacter, Lachnospira, Caecomyces, Sinimarinibacterium, Tremella,Hydrogenoanaerobacterium, Turicibacter, Clostridium XIVa, Anaerolinea,Saccharofermentans, Butyricicoccus, Olsenella, Papillibacter,Clostridium XIa, Pelotomaculum, Erysipelotrichaceae incertae sedis,Lachnospiracea incertae sedis, Solobacterium, Anaeroplasma, Ralstonia,Clostridium sensu stricto, Eubacterium, Rikenella, Lachnobacterium,Tannerella, Acholeplasma, Howardella, Selenomonas, Butyricimonas,Sharpea, Succinivibrio, Ruminobacter, Candida, Syntrophococcus,Pseudobtqrivibrio, Orpinomyces, Cyllamyces, Saccharomycetales,Phyllosticta, Ascomycota, and Piromyces.
 20. The method according toclaim 1, wherein the ruminant administered the effective amount of theruminant supplement exhibits an increase in milk production that leadsto a measured increase in milk yield.
 21. The method according to claim1, wherein the ruminant administered the effective amount of theruminant supplement exhibits an increase in milk production and improvedmilk compositional characteristics that leads to a measured increase inenergy-corrected milk.
 22. The method according to claim 1, wherein theruminant administered the effective amount of the ruminant supplementexhibits an improved milk compositional characteristic selected from thegroup consisting of: an increase in milk fat(s), an increase in milkprotein(s), an increase of carbohydrates in milk, an increase ofvitamins in milk, an increase of minerals in milk, or combinationsthereof.
 23. The method according to claim 1, wherein the ruminantadministered the effective amount of the ruminant supplement exhibits atleast a 1% increase in the average production of: milk fat(s), milkprotein(s), energy-corrected milk, or combinations thereof.
 24. Themethod according to claim 1, wherein the ruminant administered theeffective amount of the ruminant supplement exhibits at least a 10%increase in the average production of: milk fat(s), milk protein(s),energy-corrected milk, or combinations thereof.
 25. The method accordingto claim 1, wherein the ruminant administered the effective amount ofthe ruminant supplement exhibits at least a 20% increase in the averageproduction of: milk fat(s), milk protein(s), energy-corrected milk, orcombinations thereof.
 26. The method according to claim 1, wherein theruminant administered the effective amount of the ruminant supplement,further exhibits: at least one improved phenotypic trait, selected fromthe group consisting of: an improved efficiency in feed utilization,improved digestibility, an increase in polysaccharide and lignindegradation, an increase in fatty acid concentration in the rumen, pHbalance in the rumen, a reduction in methane emissions, a reduction inmanure production, improved dry matter intake, an improved efficiency ofnitrogen utilization, or combinations thereof.
 27. The method accordingto claim 1, wherein the ruminant administered the effective amount ofthe ruminant supplement, further exhibits: a shift in the microbiome ofthe rumen.
 28. The method according to claim 1, wherein the ruminantadministered the effective amount of the ruminant supplement, furtherexhibits: a shift in the microbiome of the rumen, wherein a populationof microbes present in the rumen before administration of the ruminantsupplement increase in abundance after administration of the ruminantsupplement.
 29. The method according to claim 1, wherein the ruminantadministered the effective amount of the ruminant supplement, furtherexhibits: a shift in the microbiome of the rumen, wherein a populationof microbes present in the rumen before administration of the ruminantsupplement decrease in abundance after administration of the ruminantsupplement.
 30. The method according to claim 1, wherein the ruminantadministered the effective amount of the ruminant supplement, furtherexhibits: a shift in the microbiome of the rumen, wherein a firstpopulation of microbes present in the rumen before administration of theruminant supplement increase in abundance after administration of theruminant supplement, and wherein a second population of microbes presentin the rumen before administration of the ruminant supplement decreasein abundance after administration of the ruminant supplement.